Xavier Ponsoda

Long‐term expression of differentiated functions in hepatocytes cultured in three‐dimensional collagen matrix

Hepatocytes entrapped in collagen gel and cultured in serum‐free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c‐fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin‐stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 ± 0.37 and 9 ± 2.7 nmol glucose/h/μg DNA). Collagen‐cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 ± 4 nmol urea/h/μg DNA). The rate of albumin synthesis in collagen‐entrapped cells was maintained above the day‐1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 ± 152 pmol/μg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen‐cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and gluthatione‐transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione‐transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP‐α and ‐β, and HNF‐1 and ‐4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate‐synthetase I). J Cell Physiol 177:553–562, 1998.

Evaluation of the cytotoxicity of ten chemicals on human cultured hepatocytes: Predictability of human toxicity and comparison with rodent cell culture systems

The cytotoxic effect of the first 10 chemicals on the MEIC list (evaluated in the Multicentre Evaluation of In Vitro Cytotoxicity organized by the Scandinavian Society of Cell Toxicology) was evaluated on human and rat cultured hepatocytes and in the non-hepatic murine 3T3 cell line. The MTT test was used as an endpoint to evaluate cytotoxicity after 24 hr of exposure to the chemicals. The predictability of human toxicity using human hepatocytes was analysed and compared with the results using rodent cell culture systems and rat and mouse LD(50) tests. Ferrous sulphate, diazepam and isopropyl alcohol produced about the same toxicity in all three cell culture models; paracetamol and acetylsalicylic acid were more toxic to human and rat hepatocytes than to mouse 3T3 cells; amitriptyline, ethylene glycol, methanol and ethanol were more toxic to human hepatocytes than to rodent cells. Digoxin was the most cytotoxic chemical to human hepatocytes (IC(50), 4.9 nm), the alcoholic compounds (isopropanol, ethylene glycol, ethanol and methanol) were the least toxic (IC(50), 125-819 mm) and paracetamol, acetylsalicylic acid, ferrous sulphate, diazepam and amitriptyline showed intermediate cytotoxicities (IC(50), 0.05-6 mm). The data suggest that for these 10 chemicals, acute toxicity in humans was more accurately predicted using human hepatocytes than using rat hepatocytes or mouse non-hepatic 3T3 cells.

Induction of hepatic heme oxygenase-1 by diclofenac in rodents: role of oxidative stress and cytochrome P-450 activity

BACKGROUND/AIMS The role of oxidative stress in diclofenac hepatotoxicity is still not clear. This study examined whether the drug induced heme oxygenase-1 (HO-1), a stress protein. METHODS HO-1 mRNA and HO activity were measured in mouse liver and in rat hepatocytes after treatment with diclofenac parallel to release of serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) as a marker of hepatic damage. RESULTS HO-1 was transcriptionally and dose-dependently induced by diclofenac in mouse liver and rat hepatocytes. HO-1 mRNA, ALT and SDH peaked at the same time. Mechanistic studies revealed that the drug synergized with buthionine sulfoximine (BSO) in lowering hepatic glutathione, increased the formation of reactive oxygen intermediates and activated NF-kappaB and AP-1 in rat hepatocytes. HO-1 induction and hepatic damage were increased by BSO and only HO-1 induction was attenuated by the antioxidant N-acetylcysteine. HO-1 induction was also reduced by the cytochrome P-450 inhibitors ketoconazole and tranylcypromine, concomitantly with a significant decrease in the formation of diclofenac oxidative metabolites, which may give rise to reactive compounds. CONCLUSIONS Acute treatment with diclofenac induces HO-1 in rodent hepatocytes. Induction is influenced by changes in the cellular redox states and by cytochrome P-450 activity and gives a new insight into the response of the liver to diclofenac.

Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming

During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency.

Pterostilbene-induced tumor cytotoxicity: a lysosomal membrane permeabilization-dependent mechanism.

The phenolic phytoalexin resveratrol is well known for its health-promoting and anticancer properties. Its potential benefits are, however, limited due to its low bioavailability. Pterostilbene, a natural dimethoxylated analog of resveratrol, presents higher anticancer activity than resveratrol. The mechanisms by which this polyphenol acts against cancer cells are, however, unclear. Here, we show that pterostilbene effectively inhibits cancer cell growth and stimulates apoptosis and autophagosome accumulation in cancer cells of various origins. However, these mechanisms are not determinant in cell demise. Pterostilbene promotes cancer cell death via a mechanism involving lysosomal membrane permeabilization. Different grades of susceptibility were observed among the different cancer cells depending on their lysosomal heat shock protein 70 (HSP70) content, a known stabilizer of lysosomal membranes. A375 melanoma and A549 lung cancer cells with low levels of HSP70 showed high susceptibility to pterostilbene, whereas HT29 colon and MCF7 breast cancer cells with higher levels of HSP70 were more resistant. Inhibition of HSP70 expression increased susceptibility of HT29 colon and MCF7 breast cancer cells to pterostilbene. Our data indicate that lysosomal membrane permeabilization is the main cell death pathway triggered by pterostilbene.

Diclofenac induces apoptosis in hepatocytes

Hepatotoxicity is one of the side effects associated with the administration of diclofenac, a non-steroidal anti-inflammatory drug widely used clinically. The effect of diclofenac on the early events that trigger apoptosis cascade have been evaluated in rat hepatocytes. To do this, early and late apoptotic markers, associated with the pivotal steps of the execution phase, have been evaluated after incubation with the drug. The results show that the apoptotic effect of diclofenac occurs after exposure to sub-cytotoxic concentrations of the drug (maximal non toxic concentration, MNTC, after 24-h treatment was 450 microM), without overlapping with cell necrosis (LDH leakage evaluation). Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 3 activation (3-5-fold control) was maximal after 12 h of exposure to 350 microM of the drug. The involvement of the mitochondrial permeability transition (MPT) in diclofenac-induced apoptosis was investigated. Cyclosporine A and decylubiquinone, MPT specific inhibitor, prevented the activation of caspase 3, thus showing that diclofenac opened the MPT pore. Treatment of hepatocytes with antioxidants (alpha-tocopherol, N,N-dimethylthiourea, superoxide dismutase) were able to prevent caspase cascade activation by diclofenac, revealing that oxidative stress at the mitochondrial level is involved in MPT induction. Finally, the differential cytotoxic and apoptotic effect produced in hepatocytes and non-metabolizing hepatoma cells suggest that CYP-mediated metabolism of diclofenac apoptosis may be related to the apoptotic effect of the drug.

Intracellular glutathione in human hepatocytes incubated with S-adenosyl-L-methionine and GSH-depleting drugs

The present study was undertaken to investigate (a) whether S-adenosyl-L-methionine (SAMe) added to culture medium can increase intracellular glutathione (GSH) levels in human hepatocytes and (b) whether SAMe can prevent the GSH depletion found in human hepatocytes incubated with GSH-depleting drugs (paracetamol, opiates, ethanol). Incubation of hepatocytes with increasing concentrations of SAMe resulted in a dose-dependent elevation of intracellular GSH content, which reached its maximum (35% increase) at 30 microM after 20 h. SAMe, as the only sulfur source in the medium, was efficient in repleting GSH-depleted hepatocytes following treatment with diethyl maleate. Incubation of human hepatocytes with SAMe attenuated the GSH depletion of cells incubated with toxic concentrations of paracetamol (2 mM), heroin (0.5 mM) and methadone (0.2 mM). A decrease in GSH due to exposure of hepatocytes to 50 mM ethanol was prevented when SAMe was simultaneously added to ethanol, and human hepatocytes maintained their GSH levels like non ethanol-treated cells. The experimental results of our work give the first direct evidence of the ability of exogenously administered SAMe to increase intracellular GSH levels in human hepatocytes and to prevent the GSH depletion caused by paracetamol, opiates and ethanol.

Measurement of intracellular LDH activity in 96-well cultures: A rapid and automated assay for cytotoxicity studies

The present paper describes a rapid, easy, sensitive, and automated spectrophotometric assay for intracellular lactate dehydrogenase (LDH) measurement in 96-well plates of adherent cells for cytotoxicity studies. The procedure involves “in situ” homogenization of cells, followed by measurement of LDH activity with a colorimetric method based on the reduction of a tetrazolium salt to a violet formazan by the NADH formed by LDH. Color intensity can be measured in conventional ELISA readers, and the data can be fed to an “on line” computer for rapid processing. The color absorbance measured is time- and enzyme-concentration dependent. LDH activity measured with this micromethod is coincident with that measured in larger culture plates after individual homogenization and conventional LDH measurement. The advantages of this method are the smaller number of cells required, easy automation, drastic reduction of time for processing individual wells, and the possibility of examining multiple variables in the same experiment.

Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture

The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for the correct development and functions of neurons, including intracellular traffic and signaling. In vitro ethanol exposure impairs endocytosis, exocytosis, and nucleocytoplasmic traffic in astrocytes and alters endocytosis in cultured neurons. In astrocytes, these effects relate to changes in the organization and/or function of MTs and the actin cytoskeleton. To evaluate this possibility in hippocampal cultured neurons, we analyzed if chronic ethanol exposure affects the levels, assembly, and cellular organization of both cytoskeleton elements and the possible underlying mechanisms of these effects by morphological and biochemical methods. In the experiments described below, we provide the first evidence that chronic alcohol exposure decreases the amount of both filamentous actin and polymerized tubulin in neurons and that the number of MTs in dendrites lowers in treated cells. Alcohol also diminishes the MT-associated protein-2 levels, which mainly localizes in the somatodendritic compartment in neurons. Ethanol decreases the levels of total Rac, Cdc42, and RhoA, three small guanosine triphosphatases (GTPases) involved in the organization and dynamics of the actin cytoskeleton and MTs. Yet when alcohol decreases the levels of the active forms (GTP bound) of Rac1 and Cdc42, it does not affect the active form of RhoA. We also investigated the levels of several effector and regulator molecules of these GTPases to find that alcohol induces heterogeneous results. In conclusion, our results show that MT, actin cytoskeleton organization, and Rho GTPase signaling pathways are targets for the toxic effects of ethanol in neurons.

In vitro Investigation of the Molecular Mechanisms of Hepatotoxicity

Iatrogenic Hepatitis: Intrinsic and Idiosyncratic Toxicity. Substances capable of producing liver damage and, more specifically, hepatocyte damage are known as hepatotoxins. They are classified (Zimmerman and Ishak, 1995, Castell et al., 1992) according to whether they exert their effects in all individuals, in a dose-dependent and hence predictable manner (intrinsic hepatotoxins), or in certain individuals, occasionally after several contacts, in a non-dose dependent and therefore unpredictable way (idiosyncratic hepatotoxins). These substances can act directly on cells (active hepatotoxins), or become toxic after biotransformation (latent hepatotoxins). Idiosyncratic hepatotoxicity is the consequence, either of an unusual metabolism of the drug by susceptible individuals which produce too large amounts of toxic metabolites (metabolic idiosincrasy), or is due to an immune-mediated attack to sensitised hepatocytes (drug hypersensitivity).