Jan G. Hengstler

CRYOPRESERVED PRIMARY HEPATOCYTES AS A CONSTANTLY AVAILABLE IN VITRO MODEL FOR THE EVALUATION OF HUMAN AND ANIMAL DRUG METABOLISM AND ENZYME INDUCTION

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepalocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethylsulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability ≥ 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is ≥60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.

New Hepatocyte In Vitro Systems for Drug Metabolism: Metabolic Capacity and Recommendations for Application in Basic Research and Drug Development, Standard Operation Procedures

Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4 hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this system is that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used for the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction. A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices. For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes. Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.

Human sulphotransferases are involved in the activation of aristolochic acids and are expressed in renal target tissue

Use of herbal preparations containing Aristolochia species has led to progressive nephropathy and urothelial cancer in humans. Analysis of DNA adducts formed in human target tissues and studies in animal models have pointed out a major role of the secondary plant metabolites, aristolochic acids, in these effects. Only a minority of the users of Aristolochia‐containing products developed nephropathy and cancer, suggesting differences in individual susceptibility. Differences in metabolic activation and inactivation frequently affect the susceptibility towards chemicals. Others have shown that the activation of aristolochic acids to DNA‐reactive and mutagenic metabolites requires reduction of their aryl nitro group. The biological activity of numerous nitro‐ and aminoarenes, after appropriate phase I metabolism, is strongly enhanced in the presence of acetyltransferases or sulphotransferases (SULTs). In the present study, we demonstrate that expression of human SULTs in bacterial and mammalian target cells reinforces the mutagenic activity of aristolochic acids. Using Salmonella typhimurium TA1538 as the recipient organism, we identified the expression of all 12 human SULT forms. SULT1A1 led to the strongest increase in the mutagenicity of aristolochic acids. Some activation was also observed with SULT1B1, but not with the remaining forms. The role of SULT1A1 in the activation of aristolochic acids was corroborated using S. typhimurium TA100‐ and Chinese hamster V79‐derived target cells engineered for expression of human SULT1A1 when compared with control cells. Furthermore, pentachlorophenol, an inhibitor of SULT1A1, strongly reduced the mutagenic effect of aristolochic acids in V79‐hCYP2E1‐hSULT1A1 cells. Moreover, we demonstrate that SULT1A1 and SULT1B1 are expressed in human kidney using immunoblot analysis, but their levels are substantially lower than in liver. Finally, we discuss the possibility that reactive sulphuric acid conjugates produced in other tissues are transferred to kidney and ureter.

c-kit Expression in Adenocarcinomas of the Lung

The tyrosine-kinase receptor c-kit (CD117) and its ligand stem cell factor are considered to be co-expressed in various solid tumors, including adenocarcinomas of the lung. The frequency of c-kit expression and its association with clinical parameters has not yet been evaluated in a larger population of lung adenocarcinomas. Therefore, tumor tissue of 95 consecutive patients with adenocarcinoma of the lung was stained using a polyclonal c-kit antibody. c-kit expression was correlated with relevant clinical parameters obtained by chart review. Positive c-kit expression in tumor tissue was observed in 61 of 95 patients (64%). Univariate analysis showed a significant effect of T (p = 0.003), N (p = 0.001) and M stage (p < 0.001) as well as of performance status (p = 0.001), surgical resection (p < 0.001), and LDH serum levels (p = 0.016) on survival. In contrast, c-kit protein expression was non- significant (p = 0.913). However, multivariate Cox regression with the influential parameters revealed a significant effect of c-kit expression on survival. Forward stepwise selection showed a 1.77-fold increased risk to die (hazard ratio, HR; 95% confidence interval, CI: 1.00–3.14, p = 0.047) for patients with c-kit-positive tumors. Similar data for c-kit expression were obtained by backward stepwise selection (HR: 1.78; 95% CI: 1.00–3.16; p = 0.044). In conclusion, the receptor tyrosine kinase c-kit is frequently expressed in adenocarcinomas of the lung and has a relevant effect on patient survival. The results of this study support clinical trials targeting the c-kit receptor with specific c-kit inhibitors (e.g. imatinib).

Metabolism of propafenone and verapamil by cryopreserved human, rat, mouse and dog hepatocytes: comparison with metabolism in vivo

In the present study we examined the metabolism of [14C]propafenone (P) and [14C]verapamil (V) using cryopreserved human, dog (Beagle), rat (Sprague-Dawley) and mouse (NMRI) hepatocytes. The percentage ratios of the metabolites were identified after extraction by HPLC with UV and radioactivity detection. Phase-II metabolites were cleaved using β-glucuronidase. Metabolism of the drugs by cryopreserved hepatocytes was compared with that in the respective species in vivo.All phase-I and -II metabolites known from in vivo experiments: 5-hydroxy-P (5-OH-P); 4′-hydroxy-P (4′-OH-P); N-despropyl-P (NdesP) and the respective glucuronides, were identified after incubation with cryopreserved hepatocytes. Interspecies differences were observed concerning the preferential position of propafenone hydroxylation: 5-OH-P made up 91, 51, 16 and 3% of the total metabolites after incubation with cryopreserved human (n=4), dog (n=3), rat (n=3) and mouse (n=4) hepatocytes respectively. These results are consistent with interspecies differences known from in vivo experiments. The metabolism of V is more complex than that of P. Nevertheless, all phase-I metabolites known from in vivo experiments and the expected glucuronides were identified after incubation with cryopreserved hepatocytes from all four species. As expected from the results of in vivo experiments, there were no major interspecies differences with respect to phase-I metabolites although the conjugation of verapamil phase-I metabolites by cryopreserved canine hepatocytes was much weaker than for the other species.In conclusion, phase-I and phase-II metabolism of P and V was evaluated using hepatocytes in vitro. All of the relevant interspecies differences known from in vivo experiments were identified after short-term incubation with cryopreserved hepatocytes in suspension.

ANALYSIS OF REACTIVE OXYGEN SPECIES

), Ardeystrasse 67, 44139 Dortmund, Germany Corresponding author: e-mail: stewart@ifado.de Reactive oxygen species play key roles as signalling molecules (Thannickal et al., 2000; Landar and Darley-Usmar, 2003; Pryor et al., 2006; Hengstler and Bolt, 2008) and are also involved in many patho-genic processes (Cederbaum et al., 2009; Sebai et al., 2009). Oxidative stress induc-tion is a well-documented mechanism of many non-genotoxic carcinogens (Mates et al., 2008; Hengstler and Bolt, 2007; Schug et al., 2008; Dewa et al., 2009; Binner et al., 2008). Particularly metal toxicity and car-cinogenicity often involves generation of reactive oxygen species as a key mecha-nism (Beyersmann and Hartwig, 2008; Wang et al., 2009; Cervinkova et al., 2009; Bolt and Hengstler, 2008; Verstraeten et al., 2008; Glahn et al., 2008; Pardo Andreu et al., 2009; Arivarasu et al., 2008). Given the importance of oxidative stress numerous methods have been established and optimized to reliably quantify the levels of reactive oxygen species in tissues and cells (Dikalov et al., 2007; Borgmann 2009; Setsukinai et al., 2003; Yao et al., 2004; Tarpey and Fridovich, 2001; Tarpey et al., 2004). Some of the most frequently applied techniques include: