“三明治”培养大鼠原代肝细胞模型评价P-gp介导的洛哌丁胺胆汁排泄

应用模型药物罗丹明123(Rh123)和"三明治"培养大鼠原代肝细胞(SCRH),建立并验证基于P-gp的胆汁排泄体外评价模型。SCRH培养5天后形成胆管,加入Rh123(10μmol.L-1),在含Ca2+和无Ca2+Hanks缓冲液中孵育,用LC-MS/MS分别测定Rh123的细胞蓄积量,计算胆汁排泄指数(BEI)和体外的内在胆汁清除率(CLbile,int)。在孵育体系中预先加入已知的P-gp抑制剂环孢素A(CyA)、tariquidar(TQD)和奎尼丁(QND)预孵育30 min,评价抑制P-gp转运体对底物胆汁排泄的影响。Rh123的BEI值为(17.8±1.3)%,平均CLbile,int值为(10.7±0.9)mL.min—1.kg—1,3个抑制剂对Rh123的胆汁清除均呈现剂量依赖性的抑制作用,表明模型构建成功。在此模型上,研究了P-gp介导的洛哌丁胺(LPAD)胆汁排泄以及P-gp抑制剂的影响。LPAD的BEI值为(12.9±1.2)%,平均CLbile,int为(6.1±0.3)mL.min—1.kg—1。CyA、TQD和QND对LPAD胆汁排泄也呈现浓度依赖的抑制作用,证实了P-gp转运体在LPAD胆管外排中的作用,与P-gp抑制剂合用可显著降低LPAD经胆汁的排泄。本文应用SCRH建立了B-Clear模型,为评价新药候选物基于转运体的胆汁排泄和相互作用提供了一个有用的体外工具。     

Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats.

The relationship between biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats was examined. The biliary excretion of seven model substrates in 96-h sandwich-cultured rat hepatocytes was determined by differential cumulative uptake of substrate in the monolayers preincubated in standard buffer (intact bile canaliculi) and Ca2+-free buffer (disrupted bile canaliculi). Biliary excretion in vivo was quantitated in bile duct-cannulated rats. The biliary excretion index of model substrates, equivalent to the percentage of retained substrate in the canalicular networks, was consistent with the percentage of the dose excreted in bile from in vivo experiments. The in vitro biliary clearance of inulin, salicylate, methotrexate, [D-pen2,5]enkephalin, and taurocholate, calculated as the ratio of the amount excreted into the bile canalicular networks and the area under the incubation medium concentration-time profile ( approximately 0, approximately 0, 4.1 +/- 1.0, 12.6 +/- 2.2, and 56. 2 +/- 6.0 ml/min/kg, respectively), correlated with their intrinsic in vivo biliary clearance (0.04, 0, 17.3, 34.4, and 116.9 ml/min/kg, respectively; r2 = 0.99). The model compound 264W94 was not excreted in bile either in vivo or in vitro. The glucuronide conjugate of 2169W94, the O-demethylated metabolite of 264W94, was excreted into bile in vitro when 2169W94, but not 264W94, was incubated with the monolayers; 2169W94 glucuronide undergoes extensive biliary excretion after administration of 264W94 or 2169W94 in vivo. Biliary excretion in long-term sandwich-cultured rat hepatocytes correlates with in vivo biliary excretion. The study of biliary excretion of metabolites in the hepatocyte monolayers requires consideration of the status of metabolic activities.     

小檗碱、巴马汀和药根碱在“三明治”培养大鼠原代肝细胞中的胆汁外排特征

目的研究小檗碱、巴马汀和药根碱在大鼠原代细胞的胆汁外排特征。方法建立"三明治"培养大鼠原代肝细胞(sandwich cultured rat hepatocytes,SCRH)模型,分别加入小檗碱、巴马汀、药根碱,在含Ca²⁺和无Ca²⁺缓冲液中孵育,采用UPLC-MS/MS分别测定3种生物碱的细胞蓄积量,计算胆汁排泄指数和胆汁清除率,评价小檗碱、巴马汀、药根碱在大鼠原代细胞的胆汁外排特征;并考察P-gp、Mrp2抑制剂对3种活性成分在SCRH细胞模型中的外排转运影响。结果随着孵育时间的延长,3种生物碱的细胞蓄积量增加,且含Ca²⁺条件下的细胞蓄积量与无Ca²⁺条件下相比具有明显差异;P-gp抑制剂环孢素A、维拉帕米均能减少小檗碱、巴马汀、药根碱的胆汁排泄,且呈浓度依赖性,Mrp2抑制剂MK571、丙磺舒对3种生物碱的胆汁排泄无明显影响。结论小檗碱、巴马汀、药根碱均经过胆汁外排,P-gp介导了3种生物碱的胆汁外排,Mrp2未参与其胆汁排泄。     

In vivo biliary clearance should be predicted by intrinsic biliary clearance in sandwich-cultured hepatocytes

It has been reported that in vivo biliary clearance can be predicted using sandwich-cultured rat and human hepatocytes. The predicted apparent biliary clearance (CL(bile, app)) from sandwich- cultured rat hepatocytes (SCRH) based on medium concentrations correlates to in vivo CL(bile, app) based on plasma concentrations of angiotensin II receptor blockers (ARBs), HMG-CoA reductase inhibitors (statins), β-lactam antibiotics, and topotecan. However, the predicted biliary clearance from SCRH was 7- to 300-fold lower than in vivo biliary clearance. We speculated that the process of biliary excretion might not have been evaluated using sandwich-cultured hepatocytes. To evaluate this issue, intrinsic biliary clearance (CL(bile, int)) based on intracellular compound concentrations was evaluated to investigate the in vitro-in vivo correlation of this process among ARBs, statins, β-lactam antibiotics, and topotecan. Intrinsic biliary clearance in SCRH correlated to in vivo values obtained by constant intravenous infusion of six compounds, but not rosuvastatin and cefmetazole, to rats. Moreover, differences between SCRH and in vivo CL(bile, int) (0.7-6-fold) were much smaller than those of CL(bile, app) (7-300-fold). Therefore, in vivo CL(bile, int) is more accurately reflected using SCRH than CL(bile, app). In conclusion, to predict in vivo biliary clearance more accurately, CL(bile, int) should be evaluated instead of CL(bile, app) between SCRH and in vivo.     

Alpha-naphthylisothiocyanate modulates hepatobiliary transporters in sandwich-cultured rat hepatocytes

Alpha-naphthylisothiocyanate (ANIT) induces intra-hepatic cholestasis mixed with hepatocellular injury mainly by bile ductular damage. However, its direct effect on hepatic parenchymal cells (hepatocytes) is unclear. Sandwich-cultured rat hepatocytes (SCRH) were applied to clarify this question. Though cytotoxicity was not observed (0–180 μM) in ANIT-treated SCRH, metabonomics analysis of the hepatocytes revealed a shift in the metabolic pattern and a decrease in cellular cholesterol level, accompanied by an increase in total bile acids after 48 h ANIT (5–45 μM) treatment. To assess the function of major hepatic bile acid transporters, the accumulation and efflux of [D-Pen^2,5]-enkephalin (DPDPE), 5 (and 6)-carboxy-2′,7′-dichlorofluorescein (CDF) diacetate promoiety and deuterium-labeled sodium taurocholate (d8-TCA) were measured. ANIT incubation for either 30 min or 48 h led to dose-dependent decreases in the biliary excretion index (BEI) of DPDPE and CDF, as well as the intracellular accumulation of d8-TCA, CDF and DPDPE. The basolateral efflux of d8-TCA was also decreased with its BEI barely changed. mRNA expression of multiple uptake transporters and bile acid synthesizing enzymes was down-regulated after 48 h incubation. In conclusion, ANIT could directly induce retention of bile acids in hepatocytes by inhibiting the function of bile acid transporters, which might contribute to its cholestatic effect.     

Viability assessment in sandwich-cultured rat hepatocytes after xenobiotic exposure

Troglitazone, bosentan and glibenclamide inhibit the bile salt export pump (Bsep) which transports taurocholate into bile. Sandwich-cultured rat hepatocytes maintain functional sodium taurocholate co-transporting polypeptide and Bsep transport proteins, and may be useful to study inhibition of transport by xenobiotics at concentrations below the lowest observable adverse effect level (LOAEL). The purpose of this study was to compare viability assessments determined with the neutral red, lactate dehydrogenase (LDH), alamar blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide assays in sandwich-cultured rat hepatocytes following exposure to xenobiotics known to inhibit Bsep, and to define the LOAEL for these xenobiotics in this system. The neutral red assay was not amenable to use in this model due to crystal formation on the collagen. Troglitazone decreased viability in every assay examined, with a LOAEL  100 μM. Bosentan also decreased viability as measured by the LDH, MTT and propidium iodide assays, with a LOAEL  200 μM; however, a significant decrease in viability was not observed with the alamar blue assay. Glibenclamide did not decrease viability with any assay at the xenobiotic concentrations examined in this study. Based on the results of this study, the LDH or propidium iodide assays would be the methods of choice to assess viability in sandwich-cultured rat hepatocytes after xenobiotic exposure.     

Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5-25 μM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression and total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5-30 μM) and DOX (0.025-3.0 μM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC₅₀ values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy.     

Modeling the in vitro intrinsic clearance of the slowly metabolized compound tolbutamide determined in sandwich-cultured rat hepatocytes.

An alternative approach is introduced in determining the in vitro intrinsic clearance of slowly metabolized compounds. The longterm sandwich rat hepatocyte culture was exploited, allowing for sufficient substrate depletion to obtain a reliable clearance estimation; in its physiology, it resembles the in vivo liver, thus allowing in vivo extrapolation of the in vitro clearance value. Substrate depletion of tolbutamide and the formation of its metabolites hydroxytolbutamide and carboxytolbutamide were measured in the medium and sandwich layer. Depletion data from the medium were fitted to a mathematical model incorporating system-dependent parameters (diffusion, protein binding, and partitioning) to calculate the hepatocytes' intrinsic clearance. Based on the decrease of the parent compound in the medium, a specific intrinsic clearance value, i.e., clearance per unit of volume of hepatocytes, of 0.085 min(-1) was fitted. This value was in accordance with in vivo and in vitro values from the literature. The model was verified with substrate depletion data from the sandwich layer. Data on metabolite formation showed an incomplete mass balance. A radiochemical experiment revealed the presence of three additional metabolites. These metabolites were analyzed by liquid chromatography-mass spectometry. One was identified as p-tolysulfonylurea. The structure of the other two needs to be elucidated. After the addition of these compounds to the metabolic pattern, the mass balance was completed. The in vitro clearance value was incorporated in a physiologically based pharmacokinetic literature model of tolbutamide that accurately describes the plasma concentration. The approach used in this study successfully predicts the intrinsic clearance of tolbutamide. In addition, the sandwich rat hepatocyte culture also proves to be useful in the identification of metabolic pathways.     

Role of glycosylation in trafficking of Mrp2 in sandwich-cultured rat hepatocytes

The multidrug resistance-associated protein (MRP) family plays a major role in the hepatic excretion of organic anions. The expression, localization, and function of Mrp2 (Abcc2), a canalicular multispecific organic anion transport protein, were studied in sandwich-cultured rat hepatocytes. The amount of Mrp2 protein remained constant in sandwich-cultured rat hepatocytes over 4 days in culture, but the molecular mass increased approximately 10 kDa from 190 to 200 kDa. Mrp2 was internalized initially after hepatocyte isolation and was gradually sorted to the canalicular membrane. Disposition of 5-(6)-carboxy-2',7'-dichlorofluorescein (CDF), an Mrp2 substrate, confirmed the changes in Mrp2 localization. CDF was localized predominantly inside hepatocytes at day 0 and gradually localized to the canalicular domain over time in culture. By day 4 in culture, CDF was localized exclusively in the canalicular networks. Tunicamycin, an inhibitor of glycosylation, decreased the molecular mass and simultaneously impaired the trafficking of Mrp2 to the canalicular membrane. Treatment of lysates from both day 0 (Mrp2, 190 kDa) and day 4 (Mrp2, 200 kDa) sandwich-cultured rat hepatocytes with peptide N-glycosidase F, a deglycosylation agent, resulted in a band of 180 kDa, suggesting that Mrp2 from both day 0 and day 4 was glycosylated, but Mrp2 on day 4 was more glycosylated than on day 0. In conclusion, these data support the hypothesis that glycosylation of Mrp2 is responsible for the increase in molecular mass and may be involved in directing the canalicular localization of Mrp2 in sandwich-cultured rat hepatocytes over days in culture.     

Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwich-cultured rat hepatocytes.

The purpose of the present study was to explore the utility of sandwich-cultured rat hepatocytes as an in vitro tool to examine drug interactions at the hepatic transport level. Rhodamine 123 was used as a model substrate for P-glycoprotein-mediated biliary excretion. Effects of various types of P-glycoprotein modulation on the biliary excretion index (BEI; a relative measure of the extent of biliary excretion) and the in vitro biliary clearance (CL(bile)) were determined. Significant reductions in rhodamine 123 BEI and CL(bile) were noted in the presence of the P-glycoprotein inhibitors verapamil (30-100 microM) and progesterone (100 microM). The P-glycoprotein activator quercetin (10-100 microM) enhanced rhodamine 123 CL(bile) by approximately 4-fold, with only a minor effect on BEI, suggesting that quercetin had a more pronounced effect on uptake at the basolateral membrane rather than excretion across the canalicular membrane. Treatment of hepatocytes for 48 h with dexamethasone (10 microM) resulted in significant enhancement of CL(bile), whereas rifampin (5-50 microM) increased both BEI and CL(bile), indicating that the inducing effects of dexamethasone and rifampin were occurring at the basolateral and canalicular membranes, respectively. Total rhodamine 123 uptake in sandwich-cultured rat hepatocytes was partly saturable and was affected by the presence of typical Oatp1a4 substrates (digoxin, quinine, d-verapamil, 17beta-estradiol-d-17beta-glucuronide). In summary, sandwich-cultured rat hepatocytes are a useful tool to study mechanisms of hepatobiliary drug disposition and to predict the potential for drug interactions in hepatic transport.     

Use of primary rat hepatocytes in the gel entrapment culture to predict in vivo biliary excretion

In vitro models have been widely used in characterizing the hepatobiliary elimination of compounds. However, the application of in vitro models is often limited by their imperfect simulation of in vivo situations. The current paper aims to introduce the gel entrapment culture of rat hepatocytes as an alternative method for measuring hepatobiliary transport, with the sandwich culture of rat hepatocytes set as the control. First, the culture conditions of the gel-entrapped hepatocytes were modified to enhance hepatic transport function. When cultured under optimal conditions, i.e. the collagen concentration was set to 0.6 mg/mL and the regular Williams' E medium was supplemented with epidermal growth factor, the hepatocytes maintained much higher hepatic transporter gene expression levels and transport activities than that in regular gel entrapment and sandwich culture. Compared with the actual values in rats, the predicted intrinsic biliary clearance (CL(bile,int,predicted)) of the 10 model compounds in the optimized gel entrapment culture showed a high correlation coefficient squared (R(2)) of 0.94, with the majority falling within the two-fold error range of the in vivo values, which was much better than the comparable sandwich culture. All of these results indicate that the optimized gel entrapment culture of hepatocytes is a suitable approach for estimating in vivo biliary excretion.     

Use of primary rat hepatocytes in the gel entrapment culture to predict in vivo biliary excretion

1. In vitro models have been widely used in characterizing the hepatobiliary elimination of compounds. However, the application of in vitro models is often limited by their imperfect simulation of in vivo situations. The current paper aims to introduce the gel entrapment culture of rat hepatocytes as an alternative method for measuring hepatobiliary transport, with the sandwich culture of rat hepatocytes set as the control.

2. First, the culture conditions of the gel-entrapped hepatocytes were modified to enhance hepatic transport function. When cultured under optimal conditions, i.e. the collagen concentration was set to 0.6?mg/mL and the regular Williams’ E medium was supplemented with epidermal growth factor, the hepatocytes maintained much higher hepatic transporter gene expression levels and transport activities than that in regular gel entrapment and sandwich culture.

3. Compared with the actual values in rats, the predicted intrinsic biliary clearance (CL_{bile,int,predicted}) of the 10 model compounds in the optimized gel entrapment culture showed a high correlation coefficient squared (R²) of 0.94, with the majority falling within the two-fold error range of the in vivo values, which was much better than the comparable sandwich culture.

4. All of these results indicate that the optimized gel entrapment culture of hepatocytes is a suitable approach for estimating in vivo biliary excretion.

     

Statins alter the hepatobiliary transport of unconjugated and conjugated bilirubin in sandwich-cultured rat hepatocytes

Several studies have reported that statins occasionally cause impairment of liver functions characterized by elevated serum bilirubin levels, which might be due to altered function of the multidrug resistance-associated proteins (Mrp2/3). We aimed to study the modulation of the hepatobiliary transport of bilirubin by four statin derivatives, atorvastatin, fluvastatin, pravastatin, and rosuvastatin in sandwich-cultured rat hepatocytes. All statins except pravastatin significantly inhibited the uptake of bilirubin. The biliary efflux of bilirubin conjugates was increased by pravastatin and rosuvastatin concentration dependently. Rosuvastatin stimulated not only the Mrp2 mediated biliary, but the Mrp3 mediated sinusoidal elimination, resulting in decreased intracellular bilirubin accumulation. The significantly induced Mrp2/3 protein levels (ranging from 1.5 to 1.8-fold) accounted for the elevated efflux. Cell polarization, the formation of biliary network was also significantly increased by fluvastatin, pravastatin and rosuvastatin (151%, 216% and 275% of the control, respectively). The simultaneous inhibition of the uptake and the stimulation of the sinusoidal and canalicular elimination may explain, at least in part, the clinical observation of elevated serum bilirubin levels. In conclusion, our results suggest that in spite of the elevated serum bilirubin levels, the altered Mrp2 and Mrp3 functions by statins is probably not associated with hepatotoxic effects.     

Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes.

Dexamethasone (DEX) is a well established inducer of CYP3A. These studies examined the influence of DEX treatment on transport protein expression and function in sandwich-cultured (SC) rat hepatocytes. Freshly isolated hepatocytes were cultured between two layers of gelled collagen and maintained in Dulbecco's modified Eagle's medium supplemented with DEX (0.1 microM, 0-48 h and 0.1-100 microM, 48-96 h). The expression of sinusoidal [(organic anion transporting polypeptide 1a1 (Oatp1a1), Oatp1a4, multidrug resistance-associated protein 3 (Mrp3), and Na(+)-dependent taurocholate cotransporting polypeptide (Ntcp)] and canalicular [bile salt export pump (Bsep), multidrug resistance protein 1a/b (Mdr1a/b), and Mrp2] transport proteins was determined by Western blot analysis. The accumulation and biliary excretion index (BEI; percentage of accumulated substrate in canalicular networks) of the probe substrates taurocholate (TC; 1 microM, 10 min), rhodamine 123 (Rh123; 10 microM, 30 min), and carboxy-2',7'-dichlorofluorescein (CDF; 10 microM, 10 min) were employed as measures of canalicular transport protein function in SC rat hepatocytes. DEX treatment increased CYP3A1/2, Oatp1a4, and Mrp2 expression, decreased the expression of Ntcp, and did not seem to alter the expression of Oatp1a1, Mrp3, Mdr1a/b, or Bsep. The BEI of CDF, an Mrp2 substrate, increased from 18 to 37% after DEX treatment (100 microM). The accumulation of TC, an Ntcp substrate, was reduced (<50% of control), whereas the BEI of TC, also a Bsep substrate, was unchanged. Treatment of SC rat hepatocytes with DEX resulted in alterations in the expression of CYP3A1/2 and some hepatic transport proteins. Modest alterations in hepatic transport protein function were consistent with changes in protein expression.     

Effect of interferons on P-glycoprotein-mediated rhodamine-123 efflux in cultured rat hepatocytes

The effect of interferon (IFN)-beta and IFN-gamma on P-glycoprotein (P-gp)-mediated efflux of rhodamin-123(Rho-123), a typical substrate of P-gp, was studied in rat hepatocytes in primary culture. After treatment with IFN-beta, IFN-gamma, or both for 3 days, steady-state levels of Rho-123, incorporated into the hepatocytes, were measured to evaluate the P-gp activity. Whereas IFN-beta did not affect the intracellular level of Rho-123, IFN-gamma treatment caused a significant increase of the level, suggesting that IFN-gamma treatment suppresses the expression of P-gp or its activity. A combination of the two types of IFN exhibited a similar effect to that of IFN-gamma alone. The effect of IFN-gamma was still observed in the presence of H(2)O(2), which enhances the expression and activity of P-gp. Immunoblot analysis using a monoclonal antibody C219 revealed, however, that P-gp expression was increased after treatment with IFN-gamma, but only slightly by IFN-beta treatment. These results suggest that the enhanced Rho-123 uptake of rat primary hepatocytes induced by IFN-gamma does not result from reduced expression of P-gp but, rather, from impaired maturation or dysfunction of the efflux transporter.     

Drug-induced cholestasis risk assessment in sandwich-cultured human hepatocytes

Drug-induced cholestasis (DIC) is recognized as one of the prime mechanisms for DILI. Hence, earlier detection of drug candidates with cholestatic signature is crucial. Recently, we introduced an in vitro model for DIC and evaluated its performance with several cholestatic drugs. We presently expand on the validation of this model by 14 training compounds (TCs) of the EU-EFPIA IMI project MIP-DILI.Several batches of human hepatocytes in sandwich-culture were qualified for DIC assessment by verifying the bile acid-dependent increase in sensitivity to the toxic effects of cyclosporin A. The cholestatic potential of the TCs was expressed by determining the drug-induced cholestasis index (DICI). A safety margin (SM) was calculated as the ratio of the lowest TC concentration with a DICI ≤ 0.80 to the C_max,total. Nefazodone, bosentan, perhexiline and troglitazone were flagged for cholestasis (SM < 30). The hepatotoxic (but non-cholestatic) compounds, amiodarone, diclofenac, fialuridine and ximelagatran, and all non-hepatotoxic compounds were cleared as “safe” for DIC. Tolcapone and paracetamol yielded DICI-based SM values equal to or higher than those based on cytotoxicity, thus excluding DIC as a DILI mechanism.This hepatocyte-based in vitro assay provides a unique tool for early and reliable identification of drug candidates with cholestasis risk.     

Influence of P-4502E1 induction on benzene metabolism in rat hepatocytes and on biliary metabolite excretion.

The influence of P-4502E1 induction on the metabolite pattern of benzene was studied in hepatocytes in vitro and in bile in vivo, and compared with that obtained with phenol (the major benzene metabolite). Eight metabolites from benzene and four from phenol (including conjugates) represented over 90% of total metabolites. Benzene metabolism (0.1 mM) in hepatocytes from isopropanol-treated rats (2.5 ml/kg, orally) was 3-fold higher than in corresponding cells from control rats, primarily because of increased formation of hydroquinone and phenylglutathione. Immunoblotting of microsomes revealed a parallel induction of P-4502E1 in hepatocytes from isopropanol-treated rats. In contrast, treatment with 3-methylcholanthrene or phenobarbital caused a decrease of P-4502E1, together with reduced benzene metabolism at 0.01 mM benzene. Addition of isoniazid (5 mM) resulted in a strong inhibition of benzene and phenol metabolism. Benzene metabolites were determined in bile following intraperitoneal administration of benzene (2.5 and 150 mg/kg). Biliary benzene metabolites were increased 2- to 3-fold after isopropanol treatment. Hydroquinone sulfate was identified as a major biliary metabolite of phenol. The results suggest that treatment with inducers of P-4502E1 leads, even at low benzene exposure, to an increased release of potentially myelotoxic metabolites from liver into the systemic circulation.     

Canalicular and sinusoidal disposition of bilirubin mono- and diglucuronides in sandwich-cultured human and rat primary hepatocytes.

Due to cholestasis or adverse drug effects, the excretion of bilirubin conjugates can decrease; therefore, the level of bilirubin (B) and bilirubin glucuronides (BGs) increases in the serum with the concomitant shift of bilirubin diversus monoglucuronide (BDG/BMG) equilibrium. The aim of this study was to utilize the collagen-sandwich culture of hepatocytes as an in vitro model for studying B conjugation and canalicular versus sinusoidal disposition of BGs. Canalicular and sinusoidal efflux of BMG and BDG obtained in sandwich-cultured rat primary hepatocytes was compared with that measured in human hepatocyte cultures. The BMG and BDG were separated by high-performance liquid chromatography and identified by mass spectrometry. The biliary excretion index (BEI) was estimated by measuring disposition of BGs into standard and Ca(2+), Mg(2+)-free medium. Significantly more BGs were excreted into the canalicular networks than into the medium in 96-h sandwich culture of both human and rat hepatocytes (BEI, 62.5 and 80.6, respectively). The BDG/BMG ratio in the medium versus that in the canalicular networks was 0.55/1.48, which is similar to the serum/bile values (0.6/1.5) observed in vivo by Mesa et al. [Mesa VA, De Vos R, and Fevery J (1997) J Hepatol 27:912-916]. In contrast, the BEI for p-nitrophenol glucuronide was 5.2. The low BEI value is in agreement with empirical observations, which suggest that molecules with low molecular weight are preferably excreted by the kidney. In conclusion, sandwich-cultured primary hepatocytes provide a useful in vitro method to differentiate between sinusoidal and canalicular disposition of BGs. Since the normal BDG/BMG ratio changes in hyperbilirubinemia, this model could be used to predict drug effects leading to hyperbilirubinemia.     

Comparison of coumarin-induced toxicity between sandwich-cultured primary rat hepatocytes and rats in vivo: a toxicogenomics approach.

Sandwich-cultured primary rat hepatocytes are often used as an in vitro model in toxicology and pharmacology. However, loss of liver-specific functions, in particular, the decline of cytochrome P450 (P450) enzyme activity, limits the value of this model for prediction of in vivo toxicity. In this study, we investigated whether a hepatic in vitro system with improved metabolic competence enhances the predictability for coumarin-induced in vivo toxicity by using a toxicogenomics approach. Therefore, primary rat hepatocytes were cultured in sandwich configuration in medium containing a mixture of low concentrations of P450 inducers, phenobarbital, dexamethasone, and beta-naphthoflavone. The toxicogenomics approach used enabled comparison of similar mechanistic end-points at the molecular level between in vitro and in vivo conditions, namely, compound-induced changes in multiple genes and signaling pathways. Toxicant-induced cytotoxic effects and gene expression profiles observed in hepatocytes cultured in modified medium and hepatocytes cultured in standard medium (without inducers) were compared with results from a rat in vivo study. Coumarin was used as a model compound because its toxicity depends on bioactivation by P450 enzymes. Metabolism of coumarin toward active metabolites, coumarin-induced cytotoxicity, and gene expression modulation were more pronounced in hepatocytes cultured in modified medium compared with hepatocytes cultured in standard medium. In addition, more genes and biological pathways were similarly affected by coumarin in hepatocytes cultured in modified medium and in vivo. In conclusion, these experiments showed that for coumarin-induced toxicity, sandwich-cultured hepatocytes maintained in modified medium better represent the situation in vivo compared with hepatocytes cultured in standard medium.