Wei-Bo Du, Xiao-Ping Pan, Xiao-Peng Yu, Cheng-Bo Yu, Guo-Liang Lv, Yu Chen and Lan-Juan Li

Hangzhou, China

Effects of plasma from patients with acute on chronic liver failure on function of cytochrome P450 in immortalized human hepatocytes

Wei-Bo Du, Xiao-Ping Pan, Xiao-Peng Yu, Cheng-Bo Yu, Guo-Liang Lv, Yu Chen and Lan-Juan Li

Hangzhou, China

BACKGROUND:The bioartificial liver is anticipated to be a promising alternative choice for patients with liver failure. Toxic substances which accumulate in the patients' plasma exert deleterious effects on hepatocytes in the bioreactor, and potentially reduce the efficacy of bioartificial liver devices. This study was designed to investigate the effects of plasma from patients with acute on chronic liver failure (AoCLF) on immortalized human hepatocytes in terms of cytochrome P450 gene expression, drug metabolism activity and detoxification capability.

METHODS:Immortalized human hepatocytes (HepLi-2 cells) were cultured in medium containing fetal calf serum or human plasma from three patients with AoCLF. The cytochrome P450 (CYP3A5, CYP2E1, CYP3A4) expression, drug metabolism activity and detoxification capability of HepLi-2 cells were assessed by RT-PCR, lidocaine clearance and ammonia elimination assay.

RESULTS:After incubation in medium containing AoCLF plasma for 24 hours, the cytochrome P450 mRNA expression of HepLi-2 cells was not significantly decreased compared with control culture. Ammonia elimination and lidocaine clearance assay showed that the ability of ammonia removal and drug metabolism remained stable.

CONCLUSIONS:Immortalized human hepatocytes can be exposed to AoCLF plasma for at least 24 hours with no significant reduction in the function of cytochrome P450. HepLi-2 cells appear to be effective in metabolism and detoxification and can be potentially used in the development of bioartificial liver.

(Hepatobiliary Pancreat Dis Int 2010; 9: 611-614)

acute on chronic liver failure; bioartificial liver; immortalized human hepatocyte; cytochrome P450; cell culture

Introduction

Liver failure remains a dramatic and unpredictable disease with a high mortality rate ranging from 50% to 80%.[1-3]In China, acute on chronic liver failure (AoCLF) is the most common clinical type. The bioartificial liver is anticipated to be a promising alternative for patients with liver failure as a bridge to liver transplantation or to regeneration of their own diseased liver,[4-6]in which a sufficient amount of viable hepatocytes from humans, human cell lines or pigs incorporated into bioreactors can temporarily provide the synthetic and detoxification functions of the liver.[6-8]

Many studies have demonstrated that liver failure results in the accumulation of a wide range of toxic substances within the blood[9]due to large areas of necrosis of liver tissue and acute loss of liver function. Some of these toxic substances may not only induce cytotoxicity, inhibit the regeneration of liver cells themselves, and initiate a vicious cycle with further exacerbation of liver dysfunction,[10]but also have deleterious effects on hepatocytes installed in bioreactors since many devices involve direct contact between the patient plasma and the hepatocytes, and consequently reduce the efficacy of bioartificial liver devices. Previous studies indicated that the plasma from patients with liver failure affects the growth, morphology and cellular function of hepatocytesin vitro,[11,12]including the inhibition of DNA, RNA, and protein synthesis[13,14]and apoptosis.[10,15]Cytochrome P450 is a key enzyme that contributes to metabolismin vivo; however, little is known whether plasma from patients with liver failure similarly diminishes the function of cytochrome P450 in hepatocytes.

Immortalized human liver cell lines are considered to be suitable and effective candidates for bioartificial liver devices,[16]and provide a readily available source of differentiated hepatocytes. We have currently established another immortalized human hepatocyte line, named HepLi-2, by transfection of SV40 LT into primary human hepatocytes, and these cells are potentially helpful in ammonia removal and drug metabolism. In this study, we investigated the effects of plasma from patients with AoCLF on HepLi-2 cells, in terms of cytochrome P450 gene expression, drug metabolism and detoxification, and probed the feasibility of HepLi-2 cells for future use in bioartificial liver devices.

Methods

Reagents

Dulbecco's minimum essential medium (DMEM), fetal calf serum (FCS), penicillin-streptomycin, and L-glutamine were purchased from Gibco Life Technologies, Ltd., USA. The ammonia elimination assay kit, Trizol and onestep RT-PCR reagent kit were bought from Invitrogen Co., USA. Other chemicals including lidocaine were purchased from Sigma Chemical Co., USA.

AoCLF plasma samples

Plasma samples were pooled from three patients (two males, one female; alanine transaminase: 669.18± 195.02 U/L; total bilirubin: 496.36±71.52 μmol/L; HBV DNA: positive) with HBV-related AoCLF during the course of artificial liver support system treatment which was mainly based on plasmapheresis. The samples were stored at -20 ℃ until use. The diagnosis of AoCLF was made according to the Diagnostic and Treatment Guidelines for Liver Failure, the Society of Infectious Diseases and the Society of Hepatology of the Chinese Medical Association,[17]and the position paper of the Management of Acute Liver Failure, AASLD.[1]The procedures were reviewed and approved by the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine and were also in accordance with theHelsinki Declarationof 1975 (first revision, 29th Meeting, Tokyo).

HepLi-2 cell culture

Immortalized human hepatocytes, HepLi-2 cells, were routinely cultured in plastic flasks with DMEM supplemented with 10% (vol/vol) FCS and 2 mmol/L L-glutamine. The cells were incubated at 37 ℃ in an atmosphere of 5% CO2before treatment with plasma from AoCLF patients.

Treatment of HepLi-2 cells with human plasma

After 24 hours of incubation, the HepLi-2 cells became confluent. Culture medium was replaced with DMEM containing 10% (vol/vol) FCS (control group), 10% (vol/vol) AoCLF plasma and 100% (vol/vol) AoCLF plasma. The HepLi-2 cells were re-incubated for 24 hours and then harvested for assay of cytochrome P450 gene expression. The same protocol was performed for analysis of the metabolic activity of HepLi-2 cells.

Assay of cytochrome P450 gene expression

The cytochrome P450 gene expression (CYP3A5, CYP2E1, and CYP3A4) in HepLi-2 cells was assessed by RT-PCR as described elsewhere.[18]Briefly, total cellular RNA was isolated from 5×105HepLi-2 cells using a Trizol kit. RT-PCR was performed using a one-step RT-PCR reagent kit according to the manufacturer's protocols. PCR amplification was performed with the following primers:[18]β-actin, TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA (5' primer), CTA GAA GCA TTG CGG TGG ACG ATG GAG GG (3' primer); CYP3A5, TGT CCA GCA GAA ACT GCA AA (5' primer), TTG AAG AAG TCC TTG CGT GTC (3' primer); CYP2E1, AGC ACA ACT CTG AGA TAT GG (5' primer), ATA GTC ACT GTA CTT GAA CT (3' primer); and CYP3A4, CCA AGC TAT GCT CTT CAC CG (5' primer), TCA GGC TCC ACT TAC GGT GC (3' primer).

Assay of lidocaine clearance

The cytochrome P450-dependent drug metabolism activity of HepLi-2 cells was assessed by the lidocaine clearance test as described elsewhere.[7]Briefly, after 24-hour incubation with DMEM containing different concentrations of AoCLF plasma, HepLi-2 cells were incubated with serum-free DMEM containing lidocaine (10 μg/ml) for 2 hours. Lidocaine concentration in the medium was assayed by fluorescent polarization immunoassay (Abbott Laboratories, Abbott Park, IL., USA). The tests were performed in triplicate including the controls.

Assay of ammonia elimination

The detoxification capability of HepLi-2 cells was evaluated by the ammonia elimination assay as described previously.[19,20]Briefly, 24 hours after incubation with DMEM containing different concentrations of AoCLF plasma, HepLi-2 cells were incubated with serum-free DMEM containing 1 mmol/L ammonium chloride for 4 hours. The concentration of ammonia in the medium was quantified by a commercially available ammonia assay kit. We performed all tests including the controls in triplicate.

Fig. Effects of AoCLF plasma on cytochrome P450 expression in HepLi-2 cells.

Statistical analysis

Values were presented as mean±SD. All data were analyzed with SPSS for Windows (15.0). Statistical analysis was performed using one-way ANOVA and Student'sttest. A two-tailedPvalue <0.05 was considered significant.

Results

Effects of AoCLF plasma on HepLi-2 cell cytochrome P450 expression

After 24 hours of exposure, RT-PCR showed that the mRNA expression of CYP3A5, CYP2E1, and CYP3A4 in HepLi-2 cells was almost the same in AoCLF plasma (either 10% or 100%) as in the control culture containing 10% (vol/vol) FCS (Fig.).

Effects of AoCLF plasma on HepLi-2 cell ammonia elimination

After 24-hour incubation, the ammonia elimination rate of HepLi-2 cells was 3.19±0.31 μg/106cells per hour in control culture containing 10% FCS, 3.06± 0.29 μg/106cells per hour in culture containing 10% AoCLF plasma, and 2.99±0.39 μg/106cells per hour in culture containing 100% AoCLF plasma. There was no significant difference between the three groups .

Effects of AoCLF plasma on HepLi-2 cell lidocaine clearance

After 24-hour incubation, the lidocaine clearance rate of HepLi-2 cells was 55.73±3.21 μg/108cells/ per 2 hours in control culture containing 10% FCS, 50.98± 4.72 μg/108cells per 2 hours in culture containing 10% AoCLF plasma, and 49.21±2.83 μg/108cells per hours in culture containing 100% AoCLF plasma. The difference between the three groups was not significant.

Discussion

An ideal bioartificial liver should temporarily replace or compensate for all of the functions of the diseased liver. So the metabolism and detoxification by hepatocytes intalled in the bioreactors is essential to all bioartificial liver designs. Many bioartificial liver devices, including AMC-BAL,[7]HepatAssist2000[8]and HALSS,[21]involve direct contact between the hepatocytes and the patient's plasma or blood. Thus it is important to probe the direct interactions between plasma and hepatocytes of the patients with liver failure. In this study, we analyzed the effects of AoCLF plasma on the cytochrome P450 gene expression of immortalized human hepatocytes, the HepLi-2 cell line. The results showed that the mRNA expression of cytochrome P450 was not significantly decreased in HepLi-2 cells incubated with AoCLF plasma for 24 hours as compared with the control culture. This suggested that AoCLF plasma exerted no inhibitory effect on cytochrome P450 gene expression in HepLi-2 cells.

Although the nature of the toxins and their pathogenesis are not clarified, it is clear that many toxic substances originating from either necrotic debris or agents that are normally metabolized by human hepatocytes accumulate in the patient's blood when liver failure occurs.[9,11]However, little is known about which compounds present in liver failure blood have toxic effects on hepatocytes. Hyperammonemia is common in experimental and human liver failure. Much evidence demonstrates that plasma ammonia is associated with the development of severe cerebral complications including encephalopathy and intracranial hypertension.[22]Studiesin vivoshowed that ammonia inhibits liver regeneration in animal models.[23,24]Thus, the capacity of hepatocytes to metabolize ammonia may contribute to successful bioartificial liver treatment of liver failure patients.[25]In this study, the ammonia clearance of HepLi-2 cells was not significantly decreased despite exposure to 100% AoCLF plasma for 24 hours. Furthermore, the lidocaine metabolism assay also showed that HepLi-2 cells maintained stable cytochrome P450-dependent drug metabolizing activity after 24 hours exposure to AoCLF plasma, suggesting that AoCLF plasma is not toxic to the metabolic and detoxification functions of HepLi-2 cells. These results are also consistent with a previous animal study, showing that the function of pig hepatocytes is not deteriorated when they are exposed to fulminant hepatic failure serum.[26]

In conclusion, immortalized human hepatocytes, HepLi-2 cells, can be exposed to AoCLF plasma for at least 24 hours with no significantly reduced function of cytochrome P450. HepLi-2 cells appear to be effective in metabolism and detoxification and are potentially used in the development of a bioartificial liver, but their safety requires further investigation.

Funding:This work was supported by grants from the National S&T Major Project for Infectious Disease Control of China (2008ZX10002-005), the National High Technology Research and Development Program of China (2006AA02A140), the National Natural Science Foundation of China (30630023), and Zhejiang Health Science Foundation (2009A076).

Ethical approval:The design of this study was approved by the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine.

Contributors:DWB and PXP wrote the article. All authors contributed to the design and interpretation of the study and to further drafts. LLJ is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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April 19, 2010

Accepted after revision September 28, 2010

Author Affiliations: State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Du WB, Pan XP, Yu XP, Yu CB, Lv GL, Chen Y and Li LJ)

Lan-Juan Li, MD, State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Tel: 86-571-87236759; Fax: 86-571 87236759; Email: ljli@zju.edu.cn)

? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.