Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies.

Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.

Effect of oral intake of choline-stabilized orthosilicic acid on skin, nails and hair in women with photodamaged skin.

Chronic exposure of the skin to sunlight causes damage to the underlying connective tissue with a loss of elasticity and firmness. Silicon (Si) was suggested to have an important function in the formation and maintenance of connective tissue. Choline-stabilized orthosilicic acid ("ch-OSA") is a bioavailable form of silicon which was found to increase the hydroxyproline concentration in the dermis of animals. The effect of ch-OSA on skin, nails and hair was investigated in a randomized, double blind, placebo-controlled study. Fifty women with photodamaged facial skin were administered orally during 20 weeks, 10 mg Si/day in the form of ch-OSA pellets (n=25) or a placebo (n=25). Noninvasive methods were used to evaluate skin microrelief (forearm), hydration (forearm) and mechanical anisotropy (forehead). Volunteers evaluated on a virtual analog scale (VAS, "none=0, severe=3") brittleness of hair and nails. The serum Si concentration was significantly higher after a 20-week supplementation in subjects with ch-OSA compared to the placebo group. Skin roughness parameters increased in the placebo group (Rt:+8%; Rm: +11%; Rz: +6%) but decreased in the ch-OSA group (Rt: -16%; Rm: -19%; Rz: -8%). The change in roughness from baseline was significantly different between ch-OSA and placebo groups for Rt and Rm. The difference in longitudinal and lateral shear propagation time increased after 20 weeks in the placebo group but decreased in the ch-OSA group suggesting improvement in isotropy of the skin. VAS scores for nail and hair brittleness were significantly lower after 20 weeks in the ch-OSA group compared to baseline scores. Oral intake of ch-OSA during the 20 weeks results in a significant positive effect on skin surface and skin mechanical properties, and on brittleness of hair and nails.

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.

Incorporation of ceramide 3B in dermatocosmetic emulsions: effect on the transepidermal water loss of sodium lauryl sulphate-damaged skin.

BACKGROUND: In previous work we reported on the efficacy of cosmetic body lotions enriched with skin-identical lipids to reduce the transepidermal water loss (TEWL) of ageing and sodium lauryl sulphate (SLS)-damaged skin. The observations made depended on the experimental design and clearly raised the question of the importance of the galenic formulation of skin ceramide-containing products. OBJECTIVES: The aim of the present work was to study the different galenic forms in which ceramide 3B (0.2% w/v) can be incorporated into common o/w emulsions. In addition, we investigated whether supplementation of skin care products with ceramide 3B enriched with penetration enhancers and coemulsifiers could exert a beneficial effect on barrier function, done by measuring their effects on the TEWL of SLS-induced scaly skin. RESULTS: We found that the technique of incorporating ceramide 3B into the o/w emulsions was important for their final stability. However, no additional positive effect on the TEWL values of SLS-damaged skin could be observed when the efficacy of the ceramide-containing emulsions was compared with that of proper controls. CONCLUSIONS: Although suitable galenic formulas were developed, no positive effect on TEWL could be observed when ceramide 3B was added in a final concentration of 0.2% (w/v) to different o/w emulsions and applied to SLS-damaged skin.

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Observation of hepatotoxic effects of 2-n-pentylaminoacetamide (Milacemide) in rat liver by a combined in vivo/in vitro approach.

Milacemide or 2-n-pentylaminoacetamide hydrochloride, a new glycine derivative, was found to cause elevations of plasma transaminases in patients suffering from severe depression and Alzheimer's disease. However, no signs of liver toxicity were observed during the course of earlier conducted subchronic and chronic in vivo studies in rodents and cynomolgus monkeys. In this study an in vivo/in vitro approach has been proposed to detect early alterations in key metabolic and functional liver capacities. Milacemide was administered by continuous i.v. infusion for 7 days to male Sprague-Dawley rats using subcutaneously implanted osmotic pumps. Doses were given of 0, 250 and 500 mg/kg per day. Body weight and food intake were recorded and at day 7 of exposure, Milacemide concentration, glucose, urea, triglycerides and cholesterol levels and alanine (ALT) and aspartate aminotransferase (AST) activities were measured in plasma. Non-esterified fatty acids were determined in serum. On day 8, after overnight fasting, hepatocytes were isolated. A portion of the cells derived from untreated animals (no osmotic pumps) were cultured in a primary monolayer and exposed in vitro to different Milacemide concentrations. The xenobiotic biotransformation capacity of the isolated hepatocytes was studied by measuring the cytochrome P450 content, ethoxycoumarin-O-deethylase (ECOD), pentoxyresorufin-O-deethylase (PROD), ethoxyresorufin-O-deethylase (EROD), aldrin epoxidase (AE), epoxide hydrolase (EH) and glutathione S-transferase (GST) enzyme activities. Triglycerides, cholesterol and phospholipid contents were measured on the isolated cells. At plasma concentrations of 43 and 130 microM Milacemide, the ALT activity was unchanged or significantly decreased, whereas the AST activity was increased in both cases. Other clinical chemistry parameters remained unchanged. Weight gain was significantly lower in rats treated with the high Milacemide dose. In addition, decreased food consumption was observed in all treated animals leading to significantly lower food efficiency factors for the rats treated with the high dose. Milacemide had a specific inhibitory effect on xenobiotic biotransformation: ECOD activity decreased to 60% of the control value for both Milacemide doses, PROD activity remained unaffected whereas EROD activity decreased to 65% of the control value. A decrease was also observed at the highest drug concentration for AE (to 41%), EH (to 65%), cytochrome P450 content (to 80%) and GST (to 85%). At 500 mg Milacemide kg/day, hepatocyte triglycerides levels increased 3.1-fold while cholesterol and phospholipid levels remained unaffected. Electron and light microscopy on total liver and isolated hepatocytes indicated a concentration-dependent accumulation of lipid droplets, the occurrence of numerous vacuoles in the cytoplasm and other structural abnormalities. When the cultured hepatocytes of control animals (without osmotic pumps) were exposed to Milacemide, the appearance of vacuoles and myeloid bodies could be confirmed in vitro. The results of this study using an in vivo/in vitro approach clearly show potential hepatotoxic properties of Milacemide, an effect not observed in conventional toxicity studies.

Glutathione dependent detoxication in adult rat hepatocytes under various culture conditions.

In order to obtain more information concerning the effects of culture and medium conditions on the glutathione dependent detoxication system in hepatocyte cultures, glutathione reductase (GR) and glutathione peroxidase (GPx) activities were studied in both pure cultures of adult rat hepatocytes and their co-cultures with rat epithelial cells. Cells were isolated either with an oxygen saturated Krebs Henseleit buffer (KHB) or with a non-gassed Hepes buffer. As medium conditions, additions of 10% fetal calf serum (FCS), 25 mM nicotinamide, 0.1 microM selenium and 2% dimethylsulphoxide, respectively, to the culture medium were examined. It was found that co-cultures of rat hepatocytes can cope better with oxidative stress than pure cultures do. This conclusion was reached from the following observations. When oxygenated KHB was used as isolating buffer, GR and GPx activities increased during the first days of pure culture and then slowly decreased. This was observed for all the medium conditions studied and no significant differences between the different media could be observed. For co-cultures, however, after some initial variations GR and GPx activities reached stabilized levels which were not only significantly lower than those observed for pure cultures, but were also maintained throughout the whole culture period. Supplementation of the medium had no effect on these findings with the exception of high GPx activities when Se was added to the co-culture medium. When Hepes buffer with a low oxygen content was used in cell isolation, pure cultures showed significantly lower GR and GPx activities than those first mentioned.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced and oxidized glutathione contents in adult rat hepatocytes under various culture conditions.

Reduced and oxidized glutathione contents of adult rat hepatocytes in pure culture and in co-culture with rat epithelial cells were measured under various medium conditions. To the standard medium fetal calf serum, nicotinamide, H2SeO3, dimethylsulphoxide or no supplements were added. For freshly isolated hepatocytes, intracellular contents of 24 +/- 7 nmol reduced and 0.7 +/- 0.2 nmol oxidized glutathione/mg cellular protein were obtained, respectively. In pure culture as well as in co-culture and regardless of the medium conditions involved, the protein content stays constant during the culture time with the exception of a decrease in protein content after 6 days of pure culture, caused by deterioration and loss of the hepatocytes. In both culture systems, an initial increase in intracellular reduced glutathione levels was observed, followed by a decrease and a quick normalisation in co-culture. On the contrary, in pure culture, the decrease was slower, but not transient and a stabilized situation was never reached. The various supplementations of the culture media had no significant effect on the intracellular reduced glutathione contents of both culture systems. As far as the intra- and the extracellular oxidized glutathione contents and the extracellular reduced form are concerned, these were only present in small amounts.