Evaluation of amiodarone-induced phospholipidosis by in vitro system of 3D cultured rat hepatocytes in gel entrapment

Biochemical Engineering Journal 49 (2010) 308–316

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Evaluation of amiodarone-induced phospholipidosis by in vitro system of 3D cultured rat hepatocytes in gel entrapment Chong Shen a , Guoliang Zhang b , Qin Meng a,∗ a b

Department of Chemical and Biochemical Engineering, Zhejiang University, 38 Zheda Road, Hangzhou, Zhejiang 310027, China Institute of Biological and Environmental Engineering, Zhejiang University of Technology, Zhejiang 310012, China

a r t i c l e

i n f o

Article history: Received 20 October 2009 Received in revised form 1 December 2009 Accepted 19 December 2009

Keywords: Amiodarone Phospholipidosis Hepatocytes 3D culture Gel entrapment Drug accumulation

a b s t r a c t Various mechanisms are involved in drug hepatotoxicity including reactive intermediates, steatosis, phospholipidosis, mitochondrial dysfunction, etc. Although 3D cultured hepatocytes reflected more CYP 450 mediated reactive intermediates than traditional 2D cultured hepatocytes, the 3D model has not been evaluated for drug-induced phospholipidosis. This study applied 3D cultured hepatocytes in gel entrapment for drug-induced phospholipidosis, and amiodarone was used as a model drug. By amiodarone exposure for 48 h, 3D cultured hepatocytes showed large number of lysosomal lamellar bodies (indicating phospholipidosis) and toxic response at a low dose of 2.5 ␮M, equivalent to toxic serum concentration in rats. This sensitivity to amiodarone-induced phospholipidosis might relate to the more intracellular drug distribution in 3D cultured hepatocytes. Moreover, steatosis, mitochondrial injury and oxidative stress were all sensitively detected in 3D cultured hepatocytes, well reflecting the involvement of these mechanisms in amiodarone hepatotoxicity. In addition, pretreatment of 3D cultured hepatocytes by CYP 3A1/2 inhibitor (ketoconazole) significantly reduced the toxicity of amiodarone, indicating the positive mediation of CYP 3A1/2. By comparison, 2D cultured hepatocytes did not show significant phospholipidosis and involvement of CYP 3A1/2. In conclusion, hepatocytes in 3D culture well reflected amiodarone toxicity and thus could be a promising model for phospholipidosis study. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Amiodarone is a potent antiarrhythmic drug with frequent side effects, including liver phospholipidosis and steatosis in humans [1,2] as well as animals [3,4]. The amiodarone-induced phospholipidosis is characterized by appearance of membranous lamellar bodies in lysosome [5,6] as a result of the impairment of phospholipids catabolism [7] caused by the accumulation of amiodarone in liver [8,9]. On the other hand, amiodarone inhibited ␤-oxidation of fatty acid in hepatocyte mitochondria [10] and then induced lipid accumulation in liver [11]. Subsequently, lipid peroxidation as indicated by malondialdehyde (MDA) was also detected in rats [4,12]. Moreover, as being the two major enzymes transforming amiodarone to a major metabolite of desethylamiodarone in humans

Abbreviations: CYP, cytochrome; DTNB, 5,5-dithiobis(2-nitrobenzoic acid); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathione; LDH, lactate dehydrogenase; MDA, malondialdehyde; MTT, methyl thiazolyl tetrazolium; PBS, phosphate buffer solution; ROS, reactive oxygen species; RT-PCR, reverse transcription-polymerase chain reaction; TBA, thiobarbituric acid. ∗ Corresponding author. Tel.: +86 571 87953193; fax: +86 571 87951227. E-mail address: [email protected] (Q. Meng). 1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.12.010

and rats [13,14], the cytochrome (CYP) 3A4 and 3A1 were suggested to be the most possible enzymes mediating amiodarone toxicity in vivo. Amiodarone-induced hepatotoxicity has also been investigated in vitro using hepatocytes. By 2D culture of rat hepatocyte monolayer, the amiodarone treatment in vitro was usually conducted at high concentrations (20–100 ␮M) for short incubation within 24 h [15–20]. The tested amiodarone concentration was much higher than the toxic serum concentrations of amiodarone in rats (1.62 ␮g/ml, equivalent to 2.48 ␮M) [21]. Moreover, unlike the situation in vivo, the lamellar bodies in lysosome as a typical feature of phospholipidosis were rarely observed in these studies by monolayer culture [22]. The oxidative stress was also undetectable till a high dose of 50 ␮M [17–20]. Therefore, hepatocyte monolayer seemed insensitively to reflect the amiodarone-induced hepatotoxicity in vivo. Previously, 3D cultured hepatocytes with high expression of metabolic activities showed susceptibility to CYP 450-meditated drug hepatotoxicity in spheroids [23] and gel entrapment [24,25]. However, drug-induced phospholipidosis has not been testified in 3D cultured hepatocytes. In this regard, this study would evaluate the feasibility of 3D cultured hepatocytes in gel entrapment to phospholipidosis by using amiodarone as a typical drug.

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2. Materials and methods

2.4. Effect of CYP 450 inhibitors on amiodarone-induced toxicity

2.1. Chemicals

Following 20 h of culture, hepatocytes were pretreated with fluvoxamine (5 ␮M), cimetidine (1 mM) and ketoconazole (3 ␮M) for 30 min before amiodarone treatment at 90 ␮M, while non-treated hepatocytes were incubated in the corresponding medium with ethanol (0.45%) alone. After removing the inhibitors, it was assayed by MTT reduction after hepatocytes were exposed to amiodarone at 90 ␮M for 4 h.

Williams’ E basal medium and collagenase (type IV) were purchased from Gibco (Gaithersburg, USA). Methyl thiazolyl tetrazolium (MTT), l-glutamine, penicillin and streptomycin were purchased from Amresco Inc. (Solon, Ohio, USA). Amiodarone, 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), Nile red, cimetidine, ketoconazole, insulin, dexamethasone and glucagon were purchased from Sigma (St. Louis, MO, USA). Collagen (type I) was purchased from BD Biosciences (Bedford, MA, USA). The rat albumin ELISA quantitation kit was purchased from BETHYL Laboratories, Inc. (Montgomery, TX, USA). Fetal bovine serum was obtained from Hangzhou Sijiqing Biological Eng. Material Co., Ltd. (Hangzhou, China). Rhodamine 123, dihydrorhodamine 123, RNA isolation kit, first strand cDNA synthesis kit and PCR reagents were purchased from Invitrogen (Karlsruhe, Germany). Tetraethoxypropane was a gift from Xinjing Co., Ltd. (Wuhan, China). Fluvoxamine was kindly provided by Dr. Yongzhou Hu (College of Pharmaceutical Sciences, Zhejiang University Hangzhou, China). The remaining chemicals were obtained from local chemical suppliers and were all of reagent grade. 2.2. Hepatocyte cultures All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals by the United States National Institutes of Health. Hepatocytes were isolated from male Sprague-Dawley rats (weighing 200–250 g) by the two-step collagenase perfusion method as previously described [26]. Cell viability was assessed by trypan blue exclusion and hepatocytes with a viability of greater than 85% were used. Hepatocytes were cultured in Williams’E medium supplemented with l-glutamine 2 mM, penicillin 100 U/ml, streptomycin 100 ␮g/ml, dexamethasone 100 nM, insulin 0.2 U/ml, glucagon 4 ng/ml and fetal bovine serum 5% (v/v). For the monolayer culture, each well of 24-well plates was pre-coated with 0.16 mg/l of collagen (type I). Freshly isolated rat hepatocytes were seeded at a density of 2 × 105 cells/well. The cells were cultured at 37 ◦ C in a humidified atmosphere containing 5% CO2 . For the gel entrapment culture, freshly isolated hepatocytes were mixed with the collagen solution as described before [26]. Briefly, hepatocytes were inoculated into a 3:1 (v/v) mixture of type I collagen (2.75 mg/ml) and 4-fold concentrated Williams’E medium at pH 7.4. Hepatocyte suspension at a density of 1 × 106 cells/ml was loaded into the lumen of hollow fibers with diameter of 0.6 mm and maintained in a 5% CO2 incubator for collagen gelation. Ten min later, the gel entrapped hepatocytes were extruded from the hollow fibers by 5 ml syringe into 24-well plate with a density of 1 × 105 per well. Then 1 ml prewarmed culture medium was added to each well and the plate was put into a 5% CO2 incubator for hepatocyte cultures. 2.3. Exposure of hepatocytes to amiodarone Amiodarone was dissolved into ethanol as 1000× stock solution, and then dissolved in the culture medium before use. Four hours after plating or entrapment, hepatocytes in monolayer or gel entrapment were washed with phosphate buffer solution (PBS) and then treated with amiodarone (5, 10 and 20 ␮M) for 48 h, while non-treated hepatocytes were incubated in the corresponding medium with ethanol (0.1%, v/v) alone. The culture medium was changed every 24 h before the assays were conducted.

2.5. MTT assay The MTT assay was used to evaluate the relative viability of hepatocyte in monolayer and gel entrapment. Briefly, hepatocytes were immersed in 0.65 ml of the MTT–PBS (1.15 mg/ml) in 24-well plates followed by incubation at 37 ◦ C for 3 h. After removing the MTT solution, 1.5 ml of isopropanol was added to the cells. After agitation for 1 h, the absorbance of the solution containing the extracts was read at 570 nm on a spectrophotometer as previously described [27]. 2.6. Glutathione (GSH) assay Hepatocytes in monolayer cultures were rinsed with PBS and scraped off from the wells with a rubber policeman. Gel entrapped cells were washed with PBS and then suspended in 100 ␮l of PBS. After collection, the hepatocytes were sonicated at 40 kHz and 900 W for 10 s and centrifuged at 16 000 × g to precipitate cellular fragments. The GSH content in cell supernatants was determined by DTNB assay as previously described [28]. 2.7. Reactive oxygen species (ROS) assay Accumulation of intracellular ROS was assayed by dihydrorhodamine 123. Briefly, hepatocytes (2 × 105 cells) in both monolayer and gel entrapment were incubated in PBS with 10 ␮M dihydrorhodamine 123 for 30 min at 37 ◦ C. Then, the fluorescence was determined by a fluorescence microplate reader (Tecan, Germany) with excitation at 488 nm and emission at 535 nm [29]. 2.8. Malondialdehyde (MDA) assay MDA was measured by thiobarbituric acid (TBA) reaction as described before [30]. Briefly, 2 × 105 cells in monolayer or gel entrapment were collected in tubes on ice, and then 0.3 ml phosphoric acid (0.88 mM) and 0.1 ml TBA solution (42 mM) were added, while tetraethoxypropane was used as calibrator. The tubes were tightly capped and placed in a boiling water bath for 60 min, then centrifuged at 12 000 × g for 5 min to precipitate cellular fragments. The samples were frozen at −20 ◦ C until the HPLC analyses were performed. The MDA–TBA adduct was analyzed by HPLC using an Agilent 1100 Series HPLC (Palo Alto, CA, USA) equipped with a ODS Hypersil column (250 mm × 4.6 mm, 5 ␮m particle size). The mobile phase, running at a flow-rate of 1 ml/min, comprised of methanol–phosphate buffer (0.01 mM, adjust to pH 7.0) = 65:35 and the absorbance of isolated peaks was monitored at 532 nm. 2.9. Determination of rat albumin content Albumin secreted into the culture medium by hepatocytes in monolayer and gel entrapment cultures was determined by an immunometric method (ELISA) as previously described [31].

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2.10. Assessment of mitochondrial membrane potential in hepatocytes The transmembrane potential of mitochondria was monitored fluorimetrically with a cationic dye, rhodamine 123 at the concentration of 1 ␮M. The collagen gel without cells was set as a blank for gel entrapped hepatocytes. After 20 min of incubation, the hepatocytes in monolayer and gel entrapment were respectively washed twice by PBS and fluorescence intensity was monitored at 488/535 nm of excitation/emission wavelength pair by a fluorescence microplate reader (Tecan, Germany) [32].

three times with PBS and collected in centrifuge tubes. And the gel without hepatocytes was used as the blank for gel entrapped hepatocytes. Then, 200 ␮l of acetonitrile was added and the hepatocytes were sonicated at 40 kHz and 900 W for 10 s to extract the amiodarone in hepatocytes. For the amiodarone in culture medium, the medium samples were collected and equal volume of acetonitrile was added to precipitate the proteins in medium. After extraction, the mixture was centrifuged at 16 000 × g to precipitate cellular fragments. The concentration of amiodarone in supernatant was determined by HPLC as described before [34]. 2.13. Transmission electron microscopic evaluation

2.11. Lipid accumulation in hepatocytes by Nile red staining The lipid accumulation in cultured hepatocytes was determined fluorimetrically using Nile red, a vital lipophilic dye used to label lipid droplets in the cytosol. The collagen gel without cells was set as a blank for gel entrapped hepatocytes. Both hepatocyte monolayer and gel entrapped hepatocytes were washed with PBS and then incubated for 30 min with Nile red solution at a final concentration of 3 ␮M in PBS at 37 ◦ C. Hepatocytes were washed thereafter with PBS and detected in a fluorescence microplate reader (Tecan, Germany) with excitation at 580 nm and emission at 630 nm [33].

The samples were fixed in 2.5% glutaral solution and stored at 4 ◦ C for embedding and ultrathin sectioning. The fixed samples were treated with a 1:1 (v/v) mixture of Quetol and dry ethanol for 30 min, and then followed by two treatments in Quetol for 30 and 120 min, respectively. The samples were mounted in the tip of Beem capsules, which were dried overnight in an oven at 74 ◦ C. The dried samples were sectioned on a microtome and stained with uranyl acetate for 20 min. Then they were counterstained with palladium for another 4 min, and specimens were viewed under a transmission electron microscope (JEM-1200EX, JEOL, Japan).

2.12. Assay on the concentration of amiodarone in cells and medium After incubation with 10 and 20 ␮M of amiodarone for 48 h, the hepatocytes in gel entrapment and monolayer were washed

Fig. 1. Morphology of hepatocytes in traditional 2D monolayer (A) and 3D gel entrapment (B) after 48 h of culture.

Fig. 2. Amiodarone hepatotoxicity by MTT assay (A) and rat albumin secretion (B) in hepatocyte monolayer and gel entrapped hepatocytes. The rat hepatocytes were incubated with amiodarone for 48 h, while the groups without amiodarone treatment were used as control. Data are given as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 (compared to controls).

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2.14. RT-PCR analysis Total RNA was isolated from rat hepatocytes in both monolayer and gel entrapment culture using trizol reagent. The quality of RNA was assessed by the absorbance of the samples at 260 and 280 nm. Synthesis of cDNA was performed according to the method described before [35] and amplified by the polymerase chain reaction (PCR). The gene of CYP 3A1 was measured and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. The primers are as following: CYP 3A1, 5 -CCATCCTCGTGCTCCTGTAT-3 , 5 -GGTGAAGGTGGGAGATAGCA3 (accession number AB084894), product size is 365 bp; GAPDH, 5 AAGGTCATCCCAGAGCTGAA-3 , 5 -GGATGGAATTGTGAGGGAGA-3 (accession number AB017801.1), product size is 475 bp. 2.15. Data analysis Data in this paper were expressed as relative value in percentage to the control culture without drug exposure. All values were means ± SD of three independent experiments. Comparisons between multiple groups were performed with the ANOVA test by

Fig. 4. Amiodarone-induced oxidative stress in hepatocyte monolayer and gel entrapped hepatocytes, as indicated by malondialdehyde (MDA) (A), reactive oxygen species (ROS) generation (B) and intracellular glutathione (GSH) content (C). The rat hepatocytes were incubated with amiodarone for 48 h, while the groups without amiodarone treatment were used as control. Data are given as mean ± SD (n = 3); *p < 0.05, **p < 0.01 (compared to controls). Fig. 3. Amiodarone-induced decrease of mitochondrial membrane potential (A) and lipid accumulation (B) in hepatocyte monolayer and gel entrapped hepatocytes. The mitochondrial membrane potential was detected by rhodamine 123 (RH 123) while the lipid content was assayed by Nile red staining. The rat hepatocytes were incubated with amiodarone for 48 h, while the groups without amiodarone treatment were used as control. Data are given as mean ± SD (n = 3); *p < 0.05, **p < 0.01 (compared to controls).

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Fig. 5. Ultrastructure characteristics of amiodarone-induced phospholipidosis in hepatocyte monolayer and gel entrapment after 48 h culture; (A), (B) and (C): hepatocyte monolayer with amiodarone treatment at 0, 5, 10 ␮M, respectively; (D), (E) and (F): gel entrapped hepatocytes with amiodarone treatment at 0, 5, 10 ␮M, respectively; nuclear (N), mitochrondrial (M), phospholipidosis (P) and lipid droplet (L).

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Fig. 5. (Continued ).

SPSS. P-values less than 0.05 were considered statistically significant. 3. Results 3.1. Amiodarone-induced hepatotoxicity indicated by MTT assay and liver-specific function Firstly, the morphology of hepatocytes in monolayer and gel entrapment was compared after 48 h of culture as shown in Fig. 1. The hepatocytes in monolayer culture attached on collagen-coated plates in the form of much flattened configuration (Fig. 1A), while hepatocytes in gel entrapment formed 3D structure of small aggregates or even small spheroids (Fig. 1B). The concentration-dependent hepatotoxicity of amiodarone was determined in rat hepatocytes in monolayer and gel entrapment culture by MTT assay (Fig. 2A) and albumin secretion (Fig. 2B). After 48 h of incubation, amiodarone treatment led to an obvious decrease of relative MTT value in gel entrapment culture at a dose as low as 5 ␮M (Fig. 2A). By contrast, amiodarone up to 10 ␮M induced significant cell death in hepatocyte monolayer (Fig. 2A). The EC50 of amiodarone hepatotoxicity by MTT assay in gel entrapment and monolayer was calculated as 11.7 and 14.2 ␮M, respectively. As shown in Fig. 1B, albumin secretion decreased in gel entrapment by amiodarone treatment at 2.5 ␮M, but the detectable toxic concentration was 4-fold higher in hepatocyte monolayer.

3.2. Amiodarone-induced mitochondrial damage and steatosis The mitochondrial damage was evaluated by alteration on mitochondrial membrane potential and the steatosis was reflected by Nile red staining on lipid droplet, respectively. As shown in Fig. 3A, gel entrapped hepatocytes showed mitochondrial damage at 2.5 ␮M with a reduction on mitochondrial membrane potential while this corresponding toxic dose was as high as 10 ␮M in monolayer culture. For the steatosis, only severe lipid accumulation was observed in gel entrapped hepatocytes by amiodarone treatment for 48 h (Fig. 3B). The steatosis in hepatocyte monolayer could hardly be detected in the presence of amiodarone (Fig. 3B). 3.3. Amiodarone-induced oxidative stress in hepatocytes Oxidative stress induced by amiodarone was reflected by MDA, ROS generation and GSH depletion in each culture (Fig. 4). After treatment for 48 h, 5 ␮M of amiodarone induced significant MDA generation in gel entrapped hepatocytes, while a higher concentration of amiodarone (20 ␮M) was needed in hepatocyte monolayer (Fig. 4A). Another biomarker of oxidative stress, ROS, increased by amiodarone treatment at 5 ␮M for 48 h in gel entrapped hepatocytes, while hepatocyte monolayer did not show any increase in ROS even at 20 ␮M of amiodarone treatment (Fig. 4B). Moreover, the differential effects on depletion of intracellular GSH between the two cultures were similarly presented in Fig. 4C.

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Fig. 6. The hepatocytes/culture medium concentration ratio of amiodarone in gel entrapment and monolayer. The hepatocytes were treated by 10 and 20 ␮M amiodarone for 48 h and the drug concentration in cells and culture medium was detected. Data are given as mean ± SD for each point of three separate rats. ***p < 0.001 compared to hepatocyte monolayer.

Fig. 7. Rat hepatocytes were treated by fluvoxamine (Fluv, 5 ␮M), cimetidine (Cim, 1 mM) and ketoconazole (Ket, 3 ␮M) for 30 min before amiodarone (AMD, 90 ␮M) treatment for 4 h, whereas the groups without amiodarone treatment were used as control. Data are given as mean ± SD for each point of 3 separate rats. *p < 0.05 compared to control and # p < 0.05 compared to amiodarone.

3.4. Electron microscopic evaluation of phospholipidosis Transmission electron microscope was applied for monitoring the phospholipidosis in hepatocytes (Fig. 5). As shown in Fig. 5A–C, hepatocyte monolayer only showed a few lysosomal lamellar bodies at the high amiodarone dose of 10 ␮M. In contrast, severe phospholipidosis marked by numerous formed lamellar bodies were presented in gel entrapped hepatocytes after amiodarone treatment at 5 ␮M (Fig. 5E). When the concentration increased to 10 ␮M, amiodarone induced severe mitochondrial swelling and nuclear shrinkage as well as phospholipidosis in gel entrapped hepatocytes (Fig. 5F). Without amiodarone treatment, both hepatocyte monolayer and gel entrapped hepatocytes showed no lamellar bodies or phospholipidosis (Fig. 5A and D). In addition, the cell–cell contact of gel entrapped hepatocytes was shown in Fig. 5D as most of hepatocytes formed aggregates or spheroids and the bile duct was observed in Fig. 5D-1.

Fig. 8. RT-PCR analysis for the expression of CYP 3A1. Results by RT-PCR were from hepatocytes of monolayer (M) and gel entrapment (G) after 48 h and 96 h culture.

cell viability. However, none of the CYP 450 inhibitors protected the hepatocytes in monolayer against amiodarone-induced cell death. Furthermore, the gene expression of CYP 3A1 was compared in each culture to explain the differential effects of CYP 450 inhibitors. As illustrated in Fig. 8, gene expression of CYP 3A1 maintained on a higher level within 96 h culture in gel entrapped hepatocytes but decreased rapidly in hepatocyte monolayer. Similar result was also shown in our previous study that CYP 3A1/2 activity drastically decreased within 24 h in monolayer culture while gel entrapment culture at 72 h still retained more than 70% of the initial levels [36].

3.5. Amiodarone distribution in hepatocytes and culture medium To explain the more significant phospholipidosis in gel entrapped hepatocytes, the amiodarone distribution in hepatocytes and culture medium was then investigated in each cultures for the consistency of hepatic phospholipidosis and amiodarone accumulation in liver tissue [8]. As shown in Fig. 6, the cells/medium drug distribution in gel entrapped hepatocytes was about 1.5-fold higher than that of hepatocyte monolayer after 10 and 20 ␮M of amiodarone treatment, respectively. And the distributed ratio was similar after exposure to the different concentrations at 10 and 20 ␮M in whatever gel entrapment and monolayer (Fig. 6). It should be mentioned that the drug accumulation in collagen gel was proved to be neglected after three times of washing by PBS (data not shown here). 3.6. Effect of CYP 3A1/2 in amiodarone-induced hepatotoxicity To elucidate the possible role of CYP 3A1/2 in amiodaroneinduced hepatotoxicity, rat hepatocytes were pretreated by CYP 450 inhibitors for 30 min. As indicated in Fig. 7, ketoconazole and cimetidine significantly inhibited amiodarone-induced toxicity in gel entrapped hepatocytes, while fluvoxamine failed to enhance the

4. Discussion It is well recognized that a various mechanisms involved in hepatotoxicity, including steatosis, cholestasis, phospholipidosis, reactive intermediates, mitochondria membrane dysfunction, oxidative stress, intracellular calcium homeostasis, etc. [37]. An ideal cell-based assay system is such that both the assay endpoints and toxic concentrations are more relevant to the corresponding in vivo observations [37]. In our previous study, the 3D cultured hepatocytes in gel entrapment have been developed to reflect the toxic mechanism of reactive intermediates [24,25]. The cells formed small aggregates/spheroids in gel after 48 h of culture (Fig. 1) with the structure of bile duct between two cells (Fig. 5D). Therefore, this paper aimed to evaluate its feasibility for investigating druginduced phospholipidosis. According to the analysis on MTT assay and liver-specific function (Fig. 2), the detectable toxic concentration (2.5 ␮M) in gel entrapped rat hepatocytes is very close to the toxic serum concentration of 2.48 ␮M in rats [21]. In contrast, hepatocyte monolayer only responded to amiodarone up to 10 ␮M with EC50 detected to be 14.2 ␮M, similar to the previous studies whereas the subtoxic concentration was detected to be 7.6 ␮M [22,38] and EC50

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was 14 ␮M in rat hepatocyte monolayer [39]. Moreover, significant change of other biomarkers, phospholipidosis, mitochondrial injury, lipid accumulation and oxidative stress, were all detected at low concentrations of 2.5–5 ␮M in gel entrapped hepatocytes, much lower than those in hepatocyte monolayer (Figs. 3–5). Thus, the detectable toxic concentrations in 3D cultured hepatocytes of gel entrapment were consisted well with the toxic amiodarone concentrations in vivo by various indices, while hepatocyte monolayers were insensitive to amiodarone toxicity. On the other hand, the mechanism of amiodarone toxicity in vivo was largely featured by phospholipidosis along with steatosis and CYP 450-mediated oxidative stress [40,41]. As a typical drug of phospholipidosis, amiodarone generated a large number of lysosomal lamellar bodies in rats [5,42,43], while this phospholipidosis in vivo was only reproduced in gel entrapment but not in monolayer culture in vitro (Fig. 5). As surrogate marker of steatosis, Nile red staining of lipid droplets was only largely observed in gel entrapped hepatocytes (Fig. 3B). Since amiodarone inhibited ␤-oxidation in hepatocyte mitochondria and impaired mitochondrial functions in vivo [11,44], decrease of mitochondrial membrane potential was also detected early at 2.5 ␮M of amiodarone treatment (Fig. 3A). Moreover, the oxidative stress reflected by increase on ROS/MDA together with GSH depletion was also detected in gel entrapped hepatocytes (Fig. 4), corresponding well with the results that amiodarone triggered lipid peroxidation in rats [3,4,12]. By contrast, hepatocyte monolayer was insensitive to amiodaroneinduced oxidative stress as shown in Fig. 4. Therefore, it can be concluded that 3D cultured hepatocytes in gel entrapment reflected more hepatotoxic mechanisms of amiodarone in vivo than traditional hepatocyte monolayer. To further illustrate the sensitive performance of gel entrapped hepatocytes to amiodarone hepatotoxicity, the distribution of amiodarone in hepatocytes/medium and the specific CYP 450s possibly related to amiodarone toxicity were clarified respectively. The amiodarone-induced phospholipidosis was reportedly related to its severe accumulation in liver due to the amphipathic property [8,9]. Thus, we used the hepatocyte/medium concentration ratio to represent the distribution of amiodarone in vitro. As shown in Fig. 6, the hepatocyte/medium concentration ratio was 17–19 in gel entrapped hepatocytes, which was close to the liver/plasma concentration ratio at 26 in rats after a single-dose amiodarone administration [45]. By contrast, the hepatocyte/medium concentration ratio was only 7–9 in hepatocyte monolayer (Fig. 6). The well reflected amiodarone distribution in vivo by gel entrapped hepatocytes might explain the sensitivity to amiodarone-induced phospholipidosis in Fig. 5. As the collagen gel did not accumulate amiodarone itself (data not shown here), the higher hepatocyte/medium concentration ratio in gel entrapped hepatocytes might be caused by the higher uptake to amiodarone into cells which was mediated by the higher activity of membrane transporter proteins [46], but it still needs further investigation. As desethylamiodarone, a metabolite of amiodarone by CYP 450s, also largely caused hepatotoxicity [13], several inhibitors of CYP 450s were employed to identify the specific enzymes responsible for amiodarone toxicity in vitro. As shown in Fig. 6, MTT value of rat hepatocytes in gel entrapment was significantly improved by pretreatment of ketoconazole (CYP 3A1/2 inhibitor) and cimetidine (non-selective CYP 450 inhibitor) but not by fluvoxamine (CYP 1A2 inhibitor). These results provided a support on the mediation of CYP 3A1/2 in amiodarone hepatotoxicity. However, all these inhibitors were not functional in hepatocyte monolayer (Fig. 6). Thus, the differential performance of two cultures on amiodarone hepatotoxicity might also relate to the higher maintenance of CYP 3A gene expression and activity by gel entrapment as shown in Fig. 7 and our previous study [36].

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5. Conclusion The amiodarone toxicity in rats could be reproduced in 3D cultured rat hepatocytes of gel entrapment but only some in traditional culture of hepatocyte monolayer, indicating by endpoints on phospholipidosis, steatosis, oxidative stress, mitochondrial injury and cell death. The well reflection of amiodarone toxicity might relate to the more distributed amiodarone and higher expressed CYP 3A1/2 in gel entrapped hepatocytes. Overall, gel entrapped hepatocytes could be a promising model for evaluating the hepatotoxicity of phospholipidosis drugs. Acknowledgements This research was supported by grants (no. 20576119) from NSFC (National Natural Science Foundation of China) and partly by Hi-Tech Research and Development (863) Program of China (2006AA02A140). References [1] I.S. Goldman, M.L. Winkler, S.E. Raper, M.E. Barker, E. Keung, H.I. Goldberg, T.D. Boyer, Increased hepatic density and phospholipidosis due to amiodarone, Am. J. Roentgenol. 144 (1985) 541–546. [2] J.H. Lewis, R.C. Ranard, A. Caruso, L.K. Jackson, F. Mullick, K.G. Ishak, L.B. Seeff, H.J. Zimmerman, Amiodarone hepatotoxicity: prevalence and clinicopathologic correlations among 104 patients, Hepatology 9 (1989) 679–685. [3] M. Agoston, F. Orsi, E. Feher, K. Hagymasi, Z. Orosz, A. Blazovics, J. Feher, A. Vereckei, Silymarin and vitamin E reduce amiodarone-induced lysosomal phospholipidosis in rats, Toxicology 190 (2003) 231–241. [4] J.S. Sarma, H. Pei, K. Venkataraman, Role of oxidative stress in amiodaroneinduced toxicity, J. Cardiovasc. Pharmacol. Ther. 2 (1997) 53–60. [5] M.J. Reasor, C.M. McCloud, T.L. Beard, D.C. Ebert, S. Kacew, M.F. Gardner, K.A. Aldern, K.Y. Hostetler, Comparative evaluation of amiodarone-induced phospholipidosis and drug accumulation in Fischer-344 and Sprague-Dawley rats, Toxicology 106 (1996) 139–147. [6] E.T. Baronas, J.W. Lee, C. Alden, F.Y. Hsieh, Biomarkers to monitor drug-induced phospholipidosis, Toxicol. Appl. Pharmacol. 218 (2007) 72–78. [7] K.N. Sirajudeen, P. Gurumoorthy, H. Devaraj, S.N. Devaraj, Amiodarone-induced phospholipidosis: an in vivo [14 C]-acetate uptake study in rat, Drug Chem. Toxicol. 25 (2002) 247–254. [8] M. Pirovino, U. Honegger, O. Muller, T. Zysset, A. Kupfer, M. Tinel, D. Pessayre, Differences in hepatic drug accumulation and enzyme induction after chronic amiodarone feeding of two rat strains: role of the hydroxylator phenotype? Br. J. Pharmacol. 99 (1990) 35–40. [9] R. Kannan, J.S. Sarma, M. Guha, K. Venkataraman, Tissue drug accumulation and ultrastructural changes during amiodarone administration in rats, Fundam. Appl. Toxicol. 13 (1989) 793–803. [10] B. Fromenty, C. Fisch, G. Labbe, C. Degott, D. Deschamps, A. Berson, P. Letteron, D. Pessayre, Amiodarone inhibits the mitochondrial beta-oxidation of fatty acids and produces microvesicular steatosis of the liver in mice, J. Pharmacol. Exp. Ther. 255 (1990) 1371–1376. [11] T.C. McCarthy, P.T. Pollak, E.A. Hanniman, C.J. Sinal, Disruption of hepatic lipid homeostasis in mice after amiodarone treatment is associated with peroxisome proliferator-activated receptor-alpha target gene activation, J. Pharmacol. Exp. Ther. 311 (2004) 864–873. [12] A. Vereckei, A. Blazovics, I. Gyorgy, E. Feher, M. Toth, G. Szenasi, A. Zsinka, G. Foldiak, J. Feher, The role of free radicals in the pathogenesis of amiodarone toxicity, J. Cardiovasc. Electrophysiol. 4 (1993) 161–177. [13] A. Shayeganpour, A.O. El-Kadi, D.R. Brocks, Determination of the enzyme(s) involved in the metabolism of amiodarone in liver and intestine of rat: the contribution of cytochrome P450 3A isoforms, Drug Metab. Dispos. 34 (2006) 43–50. [14] M.E. Elsherbiny, A.O. El-Kadi, D.R. Brocks, The metabolism of amiodarone by various CYP isoenzymes of human and rat, and the inhibitory influence of ketoconazole, J. Pharm. Pharm. Sci. 11 (2008) 147–159. [15] M.A. Robin, V. Descatoire, D. Pessayre, A. Berson, Steatohepatitis-inducing drugs trigger cytokeratin cross-links in hepatocytes. Possible contribution to Mallory-Denk body formation, Toxicol. In Vitro (2008). [16] N. Bhandari, D.J. Figueroa, J.W. Lawrence, D.L. Gerhold, Phospholipidosis assay in HepG2 cells and rat or rhesus hepatocytes using phospholipid probe NBD-PE, Assay Drug Develop. Technol. 6 (2008) 407–419. [17] K.M. Waldhauser, M. Torok, H.R. Ha, U. Thomet, D. Konrad, K. Brecht, F. Follath, S. Krahenbuhl, Hepatocellular toxicity and pharmacological effect of amiodarone and amiodarone derivatives, J. Pharmacol. Exp. Ther. 319 (2006) 1413–1423. [18] P. Kaufmann, M. Torok, A. Hanni, P. Roberts, R. Gasser, S. Krahenbuhl, Mechanisms of benzarone and benzbromarone-induced hepatic toxicity, Hepatology 41 (2005) 925–935. [19] R. Kikkawa, T. Yamamoto, T. Fukushima, H. Yamada, I. Horii, Investigation of a hepatotoxicity screening system in primary cell cultures – what biomark-

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