Sonja Beken

Cell morphology, albumin secretion and glutathione S-transferase expression in collagen gel sandwich and immobilization cultures of rat hepatocytes

In order to investigate whether collagen gel sandwich and immobilization cultures of rat hepatocytes are suitable in vitro models for long-term pharmaco-toxicological studies, the expression of the key phase II biotransformation enzyme, glutathione S-transferase (GST, EC 2.5.1.18), has been studied in the presence or absence of l-proline (60mug/ml) in the culture medium. Additionally, hepatocytes morphology was followed and albumin secretion into the medium measured. As judged by inverse phase light microscopy and transmission electron microscopy, cells cultured in both organotypical models remained viable and well differentiated for at least 14 days. Albumin secretion increased 2.5-fold after 7 days of culture, in comparison with the values found after 2 days, and remained thereafter relatively constant. When l-proline was added to the medium of sandwich and immobilization gel cultures, steady-state secretion levels of 7.1 and 5.1 mug albumin/hr, respectively, were already obtained after 4 days of culture. Total, Mu, Alpha and Pi class GST activities were determined using a general substrate and isoenzyme specific substrates, respectively. After 7 days of culture, total GST activities were decreased as compared with the values obtained for freshly isolated cells. On the contrary, Mu class GST activities were kept at a constant level. Alpha class GSTs were maintained at a 50% activity level and GST 7-7 activity was shown to be slightly induced. l-proline prevented an initial decline in total and Mu class GST activities in both culture models. The GST subunit pattern, measured after affinity chromatography by reversed phase HPLC, reflected the GST activity results.

Glutathione transferase activities in renal carcinomas and adjacent normal renal tissues: factors influencing renal carcinogenesis induced by xenobiotics

Abstract. In general, the biological activation of nephrocarcinogenic chlorinated hydrocarbons proceeds via conjugation with glutathione. It has mostly been assumed that the main site of initial conjugation is the liver, followed by a mandatory transfer of intermediates to the kidney. It was therefore of interest to study the enzyme activities of subgroups of glutathione transferases (GSTs) in renal cancers and the surrounding normal renal tissues of the same individuals (n=21). For genotyping the individuals with respect to known polymorphic GST isozymes the following substrates with differential specificity were used: 1-chloro-2,4-dinitrobenzene for overall GST activity (except GST θ); 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for GST α; 1,2-dichloro-4-nitro-benzene for GST µ; ethacrynic acid and 4-vinylpyridine for GST π; and methyl chloride for GST θ. In general, the normal tissues were able to metabolize the test substrates. A general decrease in individual GST enzyme activities was apparent in the course of cancerization, and in some (exceptional) cases individual activities, expressed in the normal renal tissue, were lost in the tumour tissue. The GST enzyme activities in tumours were independent of tumour stage, or the age and gender of the patients. There was little influence of known polymorphisms of GSTM1, GSTM3 and GSTP1 upon the activities towards the test substrates, whereas the influence of GSTT1 polymorphism on the activity towads methyl chloride was straightforward. In general, the present findings support the concept that the initial GST-dependent bioactivation step of nephrocarcinogenic chlorinated hydrocarbons may take place in the kidney itself. This should be a consideration in toxicokinetic modelling.

Collagen gel sandwich and immobilization cultures of rat hepatocytes: Problems encountered in expressing glutathione S-transferase activities

Collagen gel sandwich and immobilization cultures of rat hepatocytes are two recently developed organotypical culture models. Basic information with respect to the maintenance of xenobiotic biotransformation pathways and the expression of key enzyme activities, however, is lacking, making their use in pharmaco-toxicological studies rather speculative. The expression of the glutathione S-tranferase (GST; EC 2.5.1.18) activity, a key phase II enzyme, has been chosen to study the various problems that may arise in expressing the results of cytosolic enzyme activities when rat hepatocytes are cultured using both new culture models. Collagen gel matrix easily entraps culture medium proteins. These interfere with the cytosolic protein content, a parameter versus which cytosolic enzyme activities, including GSTs, are usually expressed. The following solutions are proposed: expression of the cytosolic enzyme activity results versus either (i) microsomal proteins, these are not contaminated by medium proteins, or versus (ii) cytosolic proteins after a complete collagenase digestion (0.05% collagenase type I of Sigma, 45 min, 37 degrees C) of the collagen matrix. Expression of enzyme activities versus cellular DNA appears to be unacceptable since unreliable results were obtained due to entrapped DNA in the collagen matrix. Once it was known how to express cytosolic enzyme activity, the maintenance of GST activities was investigated in both culture models using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates for total and Mu class GSTs, respectively. Two culture media were compared, control medium (DMEM) with and without supplementation of l-proline (final concentration 60 mug/ml). In both culture models, after an initial decrease, total GST activities increased significantly up to values higher than those observed for freshly isolated cells. The Mu class GST activities were maintained constant for 7 days and increased thereafter. l-Proline supplementation of the culture medium prevented the initial decline in total and Mu class GST activities in both culture configurations but did not seem to be of crucial importance in the maintenance of GST activities in both culture models.

Peroxisome mosaics in the liver of patients and the regulation of peroxisome expression in rat hepatocyte cultures

Peroxisomal deficiency disorders, which are genetically transmitted, are assumed to be expressed in all cells, and the use of cultured skin fibroblasts for diagnosis and research is based on this assumption. We describe four unrelated patients, three boys and a girl, with clinical, biochemical and microscopic evidence of a peroxisomal disorder whose livers display mosaicism, that is, parenchymal cells with peroxisomes are adjacent to cells without peroxisomes. After discussing the possible origin of these mosaics, we examine the influence of the environment on the expression of peroxisomes in adult rat hepatocytes in primary monolayer and three-dimensional culture. In this model the expression of peroxisomes varies between cells and depends upon the culture conditions.

Effects of extracellular matrix on the expression of peroxisomes in primary rat hepatocyte cultures.

BACKGROUND/AIMS Peroxisomes in wild-type cells vary between tissues and developmental stages. In the liver of some peroxisomal deficiency disorder patients, rare parenchymal cells express normal peroxisomes (mosaics); the mechanism is unknown. Our aim was to find factors regulating peroxisome expression. METHODS Liver-specific as well as peroxisome characteristics were studied in three types of primary rat hepatocyte cultures. RESULTS Total glutathione S-transferase activity and albumin secretion both increased in the collagen I sandwich and immobilization gel cultures. In contrast, in monolayers cultured on plastic, total glutathione S-transferase activity decreased and albumin secretion was only 30-40% compared to the collagen cultures. Glycogen rosettes typical of liver parenchymal cells were always abundant. Laminin and collagen IV-producing stellate cells were numerous in the monolayer but almost absent in the sandwich cultures. In 6-day-monolayer cultures, the number of liver-specific peroxisomes had decreased while atypical small or elongated peroxisomes appeared. Immunolabeling density for catalase and three beta-oxidation enzymes was decreased compared to adult rat liver; catalase specific activity in homogenates had dropped to 15% and 4% in the sandwich and monolayer cultures, respectively. In 17-day-sandwich cultures, some peroxisomes showed a very weak catalase reaction; total activity was 5%. Supplementation of the collagen type I cultures with several extracellular matrix factors could not prevent peroxisome dedifferentiation. CONCLUSION The presence of these extracellular matrix components is not sufficient for normal peroxisome expression. It is suggested that hepatocyte-specific and peroxisomal features are regulated differently. The sandwich preserves hepatocyte differentiation better than the monolayer.

Effect of epidermal growth factor in collagen gel cultures of rat hepatocytes.

Collagen gel cultures of hepatocytes represent a promising in vitro model in pharmaco-toxicology. Epidermal growth factor (EGF) is usually added to the culture medium, although one could question its value in a culture model aiming at maintaining a maximum of differentiated functional capacities. In this study, the effects of EGF (20 ng/ml) on albumin secretion, morphology and pentoxyresorufin O-depentylase (PROD), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities have been examined in both collagen gel sandwich and immobilization gel cultures of adult rat hepatocytes. Transmission electron microscopy did not show an obvious influence of epidermal growth factor (EGF) on the intracellular organization of organelles of the rat hepatocytes. It was found that EGF addition had no effect on albumin secretion in both culture models. On the contrary, the presence of EGF in the culture medium provoked in collagen gel sandwich cultures, after 7 days, significant decreases of 66% and 25% in EROD and PROD activities, respectively. On GST activities, no effect of EGF could be observed in both collagen gel cultures. Removal of EGF from the culture medium seemed to have a positive effect on the maintenance of the phase 1 biotransformation capacity of rat hepatocytes. Its addition should therefore be avoided in collagen gel cultures used in pharmaco-toxicology.

Effect of extracellular matrix composition on the expression of glutathione S-transferase isoenzymes in organotypical hepatocyte cultures.

Collagen gel sandwich and immobilization cultures of hepatocytes, using hydrated collagen type I as extracellular matrix (ECM), have been proposed as long-term in vitro models in pharmaco-toxicology. The in vivo ECM composition in the space of Disse is, however, much more complex. As a differentiated hepatocyte phenotype is thought to be highly dependent on ECM composition and biophysical characteristics, we modulated the ECM to mimic the in vivo situation. Moreover, commercially available collagen type I (Boehringer-Ingelheim) was compared to the one prepared in the laboratory from rat tails. ECM composition had no effect on albumin secretion or hepatocyte morphology in both collagen gel sandwich and immobilization cultures. Total, Alpha and Mu class GST activities in organotypical cultures with a complex or a simple collagen type I ECM were similar. The Pi class GST activity increased as a function of culture time in all culture models. Thus, mimicking the in vivo composition of the ECM did not improve the changes in GST expression that were observed in simple collagen gel cultures. The collagen type I matrix is therefore assumed to confer sufficient protection to help the hepatocytes to maintain their differentiated phenotype to a certain extent. Moreover, we hypothesize that the collagen gel matrix may act as a scaffold to keep newly synthesized ECM components in the proximity of the basolateral surfaces of the hepatocytes.

Regulatory Acceptance of Alternative Methods in the Development and Approval of Pharmaceuticals

Animal studies may be carried out to support first administration of a new medicinal product to either humans or the target animal species, or before performing clinical trials in even larger populations, or before marketing authorisation, or to control quality during production. Ethical and animal welfare considerations require that animal use is limited as much as possible. Directive 2010/63/EU on the protection of animals used for scientific purposes unambiguously fosters the application of the principle of the 3Rs when considering the choice of methods to be used.As such, today, the 3Rs are embedded in the relevant regulatory guidance both at the European (European Medicines Agency (EMA)) and (Veterinary) International Conference on Harmonization ((V)ICH) levels. With respect to non-clinical testing requirements for human medicinal products, reduction and replacement of animal testing has been achieved by the regulatory acceptance of new in vitro methods, either as pivotal, supportive or exploratory mechanistic studies. Whilst replacement of animal studies remains the ultimate goal, approaches aimed at reducing or refining animal studies have also been routinely implemented in regulatory guidelines, where applicable. The chapter provides an overview of the implementation of 3Rs in the drafting of non-clinical testing guidelines for human medicinal products at the level of the ICH. In addition, the revision of the ICH S2 guideline on genotoxicity testing and data interpretation for pharmaceuticals intended for human use is discussed as a case study.In October 2010, the EMA established a Joint ad hoc Expert Group (JEG 3Rs) with the mandate to improve and foster the application of 3Rs principles to the regulatory testing of medicinal products throughout their lifecycle. As such, a Guideline on regulatory acceptance of 3R testing approaches was drafted that defines regulatory acceptance and provides guidance on the scientific and technical criteria for regulatory acceptance of 3R testing approaches, including a process for collection of real-life data (safe harbour). Pathways for regulatory acceptance of 3R testing approaches are depicted and a new procedure for submission and evaluation of a proposal for regulatory acceptance of 3R testing approaches is described.

Modified Peroxisomes in Primary Hepatocyte Cultures

It is well known that when differentiated cells such as hepatocytes are cultured in vitro, they rapidly lose their specific features.