Stefan O. Mueller

Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor β–induced apoptosis†

Hepatocytes in culture are a valuable tool to investigate mechanisms involved in the response of the liver to cytokines. However, it is well established that hepatocytes cultured on monolayers of dried stiff collagen dedifferentiate, losing specialized liver functions. In this study, we show that hepatocyte dedifferentiation is a reversible consequence of a specific signaling network constellation triggered by the extracellular matrix. A dried stiff collagen activates focal adhesion kinase (FAK) via Src, leading to activation of the Akt and extracellular signal‐regulated kinase (ERK) 1/2 pathways. Akt causes resistance to transforming growth factor β (TGF‐β)–induced apoptosis by antagonizing p38, whereas ERK1/2 signaling opens the route to epithelial–mesenchymal transition (EMT). Apoptosis resistance is reversible by inhibiting Akt or Src, and EMT can be abrogated by blocking the ERK1/2 pathway. In contrast to stiff collagen, a softer collagen gel does not activate FAK, keeping the hepatocytes in a state where they remain sensitive to TGF‐β–induced apoptosis and do not undergo EMT. In this culture system, inhibition of p38 as well as overexpression of constitutively active Akt causes apoptosis resistance, whereas constitutively active Ras induces EMT. Finally, we show that matrix‐induced EMT is reversible by replating cells from dried stiff to soft gel collagen. Our results demonstrate that hepatocyte dedifferentiation in vitro is an active process driven by FAK‐mediated Akt and ERK1/2 signaling. This leads to similar functional and morphological alterations as observed for regenerating hepatocytes in vivo and is reversible when Akt and/or ERK1/2 signaling pathways are antagonized. Conclusion: Hepatocytes can exist in a differentiated and a dedifferentiated state that are reversible and can be switched by manipulating the responsible key factors of the signaling network. (HEPATOLOGY 2009.)

Estrogen receptors and endocrine diseases: lessons from estrogen receptor knockout mice

The estrogen receptors ERalpha and ERbeta are the main mediators of estrogen action and estrogens play an important role in a variety of aspects of physiology besides their well acknowledged function in reproduction. In vivo and in vitro studies indicate that the estrogen receptors are mechanistically implicated in endocrine-related diseases. Recent studies with estrogen receptor knockout mice have helped to unravel the role of the estrogen receptors in brain degeneration, osteoporosis, cardiovascular diseases and obesity.

Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress.

High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the nephrotoxin Cyclosporine A (CsA) at therapeutic and supratherapeutic concentrations for 14days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15μM induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein-response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5μM CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress.

Activation of estrogen receptor α and ERβ by 4-methylbenzylidene-camphor in human and rat cells: comparison with phyto- and xenoestrogens

4-Methylbenzylidene-camphor (4-MBC) is an organic sunscreen that protects against UV radiation and may therefore help in the prevention of skin cancer. Recent results on the estrogenicity of 4-MBC have raised concerns about a potential of 4-MBC to act as an endocrine disruptor. Here, we investigated the direct interaction of 4-MBC with estrogen receptor (ER) alpha and ERbeta in a series of studies including receptor binding, ER transactivation and functional tests in human and rat cells. 4-MBC induced alkaline phosphatase activity, a surrogate marker for estrogenic activity, in human endometrial Ishikawa cells. Interestingly, 4-MBC induced weakly ERalpha and with a higher potency ERbeta mediated transactivation in Ishikawa cells at doses more than 1 microM, but showed no distinct binding affinity to ERalpha or ERbeta. In addition, 4-MBC was an effective antagonist for ERalpha and ERbeta. In an attempt to put 4-MBCs estrogenic activity into perspective we compared binding affinity and potency to activate ER with phyto- and xenoestrogens. 4-MBC showed lower estrogenic potency than genistein, coumestrol, resveratrol, bisphenol A and also camphor. Analysis of a potential metabolic activation of 4-MBC that could account for 4-MBCs more distinct estrogenic effects observed in vivo revealed that no estrogenic metabolites of 4-MBC are formed in primary rat or human hepatocytes. In conclusion, we were able to show that 4-MBC is able to induce ERalpha and ERbeta activity. However, for a hazard assessment of 4-MBCs estrogenic effects, the very high doses of 4-MBC required to elicit the reported effects, its anti-estrogenic properties as well as its low estrogenic potency compared to phytoestrogens and camphor has to be taken into account.

Overview of in vitro tools to assess the estrogenic and antiestrogenic activity of phytoestrogens

There is an intense discussion in the scientific and even more so in the public community as well as regulatory agencies about the potential benefits or detrimental effects of plant-derived compounds that may affect the endocrine system, especially estrogen signaling pathways. These so-called phytoestrogens are found in the normal western diet and predominantly in an eastern or soy-based diet and the potency of the isolated compounds to interact with the known receptors for estrogen varies tremendously. The estrogen receptors, ER alpha and ER beta, mediate the effects of endogenous estrogens, i.e. regulation of reproductive function, tissue development, cell proliferation and differentiation. In this review, in vitro test systems available to date for the screening of estrogenic and antiestrogenic activity including mechanism-based assays are described. The potency of phytoestrogens determined using these in vitro assays are compared with the potency of endogenous estrogens and results obtained in vitro are compared with effects in vivo. Finally, the impact of in vitro assays to determine estrogenicity on human hazard assessment is discussed as well as other non ER-mediated mechanisms that may contribute to potential beneficial or adverse effects of phytoestrogens in man.

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: a valuable model for in vitro toxicity and drug-drug interaction studies.

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug-drug interaction studies.

Species-specific toxicity of diclofenac and troglitazone in primary human and rat hepatocytes.

Troglitazone was withdrawn from the market shortly after approval for diabetes type II therapy because of strong hepatotoxic effects in man that could not be predicted from regulatory animal or in vitro studies. Another pharmaceutical that is regularly associated with adverse effects on the liver, sometimes leading to acute liver failure, is the widely used non-steroidal anti-inflammatory drug (NSAID) diclofenac. Since the underlying molecular mechanisms are not yet fully known, we treated primary rat and human hepatocyte monolayer cultures for 24h with different doses of troglitazone and diclofenac to analyze species differences related to toxicity in vitro. Metformin an antidiabetic drug which does not cause severe adverse reactions served as negative control. Human hepatocytes showed a higher sensitivity to troglitazone than rat hepatocytes, while diclofenac-induced cytotoxicity at fairly similar concentrations. By co-treatment with specific inhibitors for cytochrome P450 (CYP) 2C and CYP3A - the major phase I enzymes involved in liver xenobiotic metabolism - we could confirm the prominent role of CYP3A in the bioactivation of troglitazone as well as the role of CYP3A and CYP2C in the activation of diclofenac. Inhibition of these enzymes increased the viability of treated cells in both species. Furthermore, we were able to demonstrate marked species differences in gene expression patterns of troglitazone treated rat and human hepatocytes. In contrast to rat hepatocytes, human cells showed distinct upregulation of various CYPs, regulators of xenobiotic metabolism and marker genes for oxidative stress. In contrast, gene expression alterations in rat and human hepatocytes treated with Diclofenac were rather similar. Altogether our study showed that species-specific effects as well as indications for the mode of action of compounds can be addressed by the use of primary hepatocyte cultures from various species in combination with gene expression profiling.

Development of a New Screening Assay to Identify Proteratogenic Substances using Zebrafish Danio rerio Embryo Combined with an Exogenous Mammalian Metabolic Activation System (mDarT)

The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

Primary hepatocytes as a model to analyze species-specific toxicity and drug metabolism

Background: Compound failures have been emerging in later stages of pharmaceutical drug development and are in many cases not detected until the administration to humans in clinical trials or even after approval. Among the most frequent adverse effects are drug-induced liver injury generated by species-specific susceptibilities (e.g., in xenobiotic metabolism and/or the occurrence of idiosyncratic drug hepatotoxicity). Objectives: Detecting or predicting unfavorable drug effects on the liver as early as possible in drug development is crucial in making the drug development process more efficient and the application of new drugs to humans in clinical studies and medical use safer. Methods: To achieve this goal, primary hepatocyte cultures from various species, including humans, are analyzed for morphological, functional and gene expression alterations after compound treatment. Results/conclusion: Primary hepatocyte cultures appear to be a promising tool for the detection of general or liver toxicity and the evaluation of species-specific drug effects.

Oestrogen receptors pathways to oestrogen responsive elements: The transactivation function-1 acts as the keystone of oestrogen receptor (ER)β-mediated transcriptional repression of ERα

Oestrogen receptors (ER)alpha and beta modify the expression of genes involved in cell growth, proliferation and differentiation through binding to oestrogen response elements (EREs) located in a number of gene promoters. Transient transfection of different luciferase reporter vectors 3xEREs-Vit, 2xEREs-tk and ERE-C3 showed that the transactivation capacity of both ER subtypes was influenced by 1) the nature of the inducer (oestradiol (E2), phyto- and anti-oestrogen (AE)), 2) the structure of the promoter (nucleotidic sequence, number of ERE, length of the promoter sequence) and 3) the cell line (containing endogenous ER (MCF-7) or in which ER was stably expressed (MDA-MB-231-HE-5 (ERalpha+) or MDA-MB-231-HERB (ERbeta+)). ER subtype did not display the same efficacy on the different constructions in the presence of E2 and of AE according to the cell (e.g. in MCF-7 cells: tk>>Vit>>C3 approximately 0 while in MDA-MB-231 cells: Vit>>tk approximately C3). E2 response was higher in MCF-7 cells, probably due to higher ER expression level (maximal at 10(-10)M instead of 10(-8)M for E2 in HE-5 cells). Finally, the same ligand could exert opposite activities on the same promoter according to the ER isoform expressed: in the MDA-MB-231 cells, AE acted as inducers of the C3 promoter via ERbeta whereas ERalpha/AE complexes down-regulated this promoter. Approximately 70% of breast tumours express ER and most tumour cells coexpress both ER isotypes. Thus, different types of ER dimers can be formed in such tumours (ERbeta or ERalpha homodimers or ERalpha/ERbeta heterodimers). We therefore studied the influence of the coexistence of the two ERs on the ligand-induced transcriptional process following transient transfection of ERalpha in ERbeta+ cells, and inversely ERbeta in ERalpha+ cells. ERbeta-transfection inhibited the E2- and genistein-induced ERalpha-dependent transcription on all promoters in all cell lines except C3 in MCF-7; this inhibitory effect was lost following transfection of ERbeta deleted of its AF-1 (ERbeta-AF-2). These results suggest that the dominant negative properties of ERbeta are mainly due to its AF-1 function. Interestingly, transfection of an ERbeta-AF-2 construct into MCF-7 cells potentiated the transcription inhibitory capacity of 4-OH-tamoxifen (OHT) on the Vit and tk promoters. Thus, (1) OHT exerts an agonistic activity through the AF-1 function of ER and (2) expression of ERbeta in breast cancer cells seems to favour the AE treatment. Contrary to ERbeta, ERalpha-transfection had little effect on ERbeta transactivation capacity in HERB cells. Finally, the ratio ERalpha/ERbeta constitutes one decisive parameters to orientate the transcriptional mechanism of a target gene in the presence of agonist as well as of antagonist ligands.