Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI.

Drug induced liver injury (DILI) is an idiosyncratic adverse drug reaction leading to severe liver damage. Kupffer cells (KC) sense hepatic tissue stress/damage and therefore could be a tool for the estimation of consequent effects associated with DILI. Aim of the present study was to establish a human in vitro liver model for the investigation of immune-mediated signaling in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver specimens. The isolated KC yield was 1.2 ± 0.9 × 10(6) cells/g liver tissue with a purity of >80%. KC activation was investigated by the measurement of reactive oxygen intermediates (ROI, DCF assay) and cell activity (XTT assay). The initial KC activation levels showed broad donor variability. Additional activation of KC using supernatants of hepatocytes treated with hepatotoxic drugs increased KC activity and led to donor-dependent changes in the formation of ROI compared to KC incubated with supernatants from untreated hepatocytes. Additionally, a compound- and donor-dependent increase in proinflammatory cytokines or in anti-inflammatory cytokines was detected. In conclusion, KC related immune signaling in hepatotoxicity was successfully determined in a newly established in vitro liver model. KC were able to detect hepatocyte stress/damage and to transmit a donor- and compound-dependent immune response via cytokine production.

Age-dependent changes of the antioxidant system in rat livers are accompanied by altered MAPK activation and a decline in motor signaling.

Aging is characterized by a progressive decrease of cellular functions, because cells gradually lose their capacity to respond to injury. Increased oxidative stress is considered to be one of the major contributors to age-related changes in all organs including the liver. Our study has focused on elucidating whether important antioxidative enzymes, the mTOR pathway, and MAPKs exhibit age-dependent changes in the liver of rats during aging. We found an age-dependent increase of GSH in the cytosol and mitochondria. The aged liver showed an increased SOD enzyme activity, while the CAT enzyme activity decreased. HO-1 and NOS-2 gene expression was lower in adult rats, but up-regulated in aged rats. Western blot analysis revealed that SOD1, SOD2, GPx, GR, γ-GCL, and GSS were age-dependent up-regulated, while CAT remained constant. We also demonstrated that the phosphorylation of Akt, JNK, p38, and TSC2(Ser1254) decreased while ERK1/2 and TSC2(Thr1462) increased age-dependently. Furthermore, our data show that the mTOR pathway seems to be activated in livers of aged rats, and hence stimulating cell proliferation/regeneration, as confirmed by an age-dependent increase of PCNA and p-eIF4E(Ser209) protein expression. Our data may help to explain the fact that liver cells only proliferate in cases of necessity, like injury and damage. In summary, we have demonstrated that, age-dependent changes of the antioxidant system and stress-related signaling pathways occur in the livers of rats, which may help to better understand organ aging.

3D Cultivation Techniques for Primary Human Hepatocytes.

One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

Darbepoetin inhibits proliferation of hepatic cancer cells in the presence of TGF-ß.

Darbepoetin (DPO), an erythropoietin (EPO) derivative, was licensed in 2002 to treat patients with solid tumors suffering from chemotherapy-dependent anemia, although various tumors express EPO to improve vascularization, thus favoring tumor growth and spreading. Therefore, we wanted to investigate direct effects of DPO on the liver tumor cell lines HepG2, SkHep1, Huh-7, AKN1, HCC-T and HCC-M, as well as on primary human hepatocytes (hHeps). DPO (0-40 ng/ml) did not affect viability of hHeps, HepG2, SkHep1, AKN1, HCC-T and HCC-M cells, as determined by Resazurin conversion. However, Huh-7 cells' viability dose-dependently decreased from 5 ng/ml DPO on. Lack of LDH release into culture medium and negative DNA laddering excluded apoptosis or necrosis as the cause for the reduced Resazurin conversion. In Huh-7 cells, DPO increased the expression of p53. Interestingly, Huh-7 cells showed the highest basal TGF-ß1 expression as compared to the other cell lines. Upon inhibition of TGF-ß1 signaling, DPO no longer reduced viability in Huh-7 cells. On the contrary, co-incubation with TGF-ß1 made the other cell lines responsive to DPO. Summarizing our data, we show that DPO reduces the growth of Huh-7 cells by up-regulation of the tumor-suppressor gene p53. This mechanism seems to be dependent on a strong TGF-ß expression and corresponding signaling in these cells, as other cell lines became responsive to DPO with TGF-ß1 supplementation. The knowledge of this mechanism offers great perspectives for the understanding and treatment of solid liver tumors.

Long-term culture of primary hepatocytes: new matrices and microfluidic devices.

Prediction of in vivo drug-induced hepatotoxicity by in vitro cell culture systems is still one of the main challenges in drug development. To date, most in vitro approaches are based on monolayer cultures of primary hepatocytes, although it is known that they rapidly lose their morphology and liver-specific functions, such as activities of drug-metabolizing enzymes and transporters. Hepatocyte dedifferentiation can be delayed by culturing cells in a 3D environment. Combination with continuous medium flow, which creates a more physiological situation, further improves the maintenance of hepatic functions. Here, we present recently developed hydrogels and scaffolds for 3D culture of hepatocytes, which aim at preserving hepatic morphology and functionality for up to 4 weeks in culture. Furthermore, major benefits and drawbacks of microfluidic devices for in vitro hepatotoxicity screening are discussed. Although promising advances have been made regarding the preservation of hepatic functions in 3D flow culture, major issues, such as expensive equipment, large cell numbers and low throughput, are still hampering their use in drug toxicity screening. For these devices to be applied and accepted in the drug-developing industry, it is necessary to combine easily accessible matrices that highly preserve the activities of drug-metabolizing enzymes with a user-friendly microfluidic platform, thereby finding the right balance between reflecting the in vivo situation and enabling satisfying throughput for drug candidate screening.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Human parenchymal and non-parenchymal liver cell isolation, culture and characterization.

Many reports describing parenchymal liver cell isolation have been published so far. However, recent evidence has clearly demonstrated that non-parenchymal liver cells play an important role in many pathophysiologies of the liver, such as drug-induced liver diseases, inflammation, and the development of liver fibrosis and cirrhosis. In this study, we present an overview of the current methods for isolating and characterizing parenchymal and non-parenchymal liver cells.

Alternaria toxins: Altertoxin II is a much stronger mutagen and DNA strand breaking mycotoxin than alternariol and its methyl ether in cultured mammalian cells.

Altertoxin II (ATX II) is one of the several mycotoxins produced by Alternaria fungi. It has a perylene quinone structure and is highly mutagenic in Ames Salmonella typhimurium, but its mutagenicity in mammalian cells has not been studied before. Here we report that ATX II is a potent mutagen in cultured Chinese hamster V79 cells, inducing a concentration-dependent increase of mutations at the hypoxanthine guanine phosphoribosyltransferase gene locus at concentrations similar to that of the established mutagen 4-quinoline-N-oxide. Thus, ATX II is at least 50-times more potent as a mutagen than the common Alternaria toxins alternariol (AOH) and alternariol methyl ether (AME). In contrast to AOH and AME, ATX II does not affect the cell cycle of V79 cells. ATX II also causes DNA strand breaks in V79 cells, with a potency again exceeding that of AOH and AME. The high mutagenic and DNA strand breaking activity of ATX II raises the question of whether this Alternaria toxin poses a risk for public health, and warrants studies on the occurrence of ATX II and other perylene quinone-type mycotoxins in food and feed.

Identification of a polyketide synthase required for alternariol (AOH) and alternariol-9-methyl ether (AME) formation in Alternaria alternata.

Alternaria alternata produces more than 60 secondary metabolites, among which alternariol (AOH) and alternariol-9-methyl ether (AME) are important mycotoxins. Whereas the toxicology of these two polyketide-based compounds has been studied, nothing is known about the genetics of their biosynthesis. One of the postulated core enzymes in the biosynthesis of AOH and AME is polyketide synthase (PKS). In a draft genome sequence of A. alternata we identified 10 putative PKS-encoding genes. The timing of the expression of two PKS genes, pksJ and pksH, correlated with the production of AOH and AME. The PksJ and PksH proteins are predicted to be 2222 and 2821 amino acids in length, respectively. They are both iterative type I reducing polyketide synthases. PksJ harbors a peroxisomal targeting sequence at the C-terminus, suggesting that the biosynthesis occurs at least partly in these organelles. In the vicinity of pksJ we found a transcriptional regulator, altR, involved in pksJ induction and a putative methyl transferase, possibly responsible for AME formation. Downregulation of pksJ and altR caused a large decrease of alternariol formation, suggesting that PksJ is the polyketide synthase required for the postulated Claisen condensations during the biosynthesis. No other enzymes appeared to be required. PksH downregulation affected pksJ expression and thus caused an indirect effect on AOH production.

The Alternaria mycotoxins alternariol and alternariol methyl ether induce cytochrome P450 1A1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor.

The Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME) are potential carcinogens. As planar compounds, AOH and AME are preferentially metabolized by cytochrome P450 (CYP) 1A1 and 1A2. The most prominent regulator of CYP1A1 is the dimeric transcription factor complex AhR/ARNT, which is activated by planar ligands. Therefore, we studied the activation of AhR/ARNT by AOH and AME and monitored CYP1A1 induction in murine hepatoma cells (Hepa-1c1c7). Indeed, AOH and AME enhanced the levels of CYP1A1 in Hepa-1c1c7 cells but not in cells with inactivated AhR (Hepa-1c1c12) or ARNT (Hepa-1c1c4). AOH and AME did not increase the production of reactive oxygen species but reduced cell counts in Hepa-1c1c7 cells after 24 and 48 h. This effect, however, was independent of AhR/ARNT. At 48 h, AOH and AME increased apoptosis dependent on AhR and ARNT. In conclusion, AOH and AME are novel inducers of the AhR/ARNT pathway, which mediates induction of CYP1A1 and apoptosis and might thereby contribute to the toxicity of these mycotoxins.

Oxidative metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in precision-cut rat liver slices in vitro.

SCOPE: Monohydroxylation of alternariol (AOH) and alternariol-9-methyl ether (AME) has previously been reported as a prominent metabolic route under cell-free conditions. This pathway gives rise to several catechol metabolites and may therefore be of toxicological relevance. METHODS AND RESULTS: To clarify whether hydroxylation of AOH and AME occurs under in vivo-like conditions in the presence of conjugation reactions, the metabolism of the Alternaria toxins has now been studied in precision-cut rat liver slices. Four catechol metabolites of AOH and two of AME, together with several of their O-methylation products, as catalyzed by catechol-O-methyl transferase, were clearly identified after incubation of the liver slices with AOH and AME. These metabolites were predominantly present as conjugates with glucuronic acid and/or sulfate. In preliminary studies with bile duct-cannulated male rats dosed with AOH by gavage, the four monohydroxylated metabolites of AOH could also be demonstrated in the bile either as catechols or as O-methyl ethers. CONCLUSION: These experiments clearly show that AOH and AME undergo catechol formation in vivo and warrant closer examination of the toxicological significance of this metabolic pathway.

Absorption and metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in Caco-2 cells in vitro.

Alternariol (AOH) and alternariol-9-methyl ether (AME) are major toxins produced by fungi of the genus Alternaria. In order to simulate their in vivo intestinal absorption and metabolism, AOH and AME have been studied in differentiated Caco-2 cells and in the Caco-2 Millicell® system in vitro. AOH was found to be readily conjugated to two glucuronides and one sulfate, whereas AME gave rise to one major glucuronide and one sulfate. Whereas the glucuronides of AOH and AME were sequestered about equally well into the basolateral and the apical compartment, the sulfates of both toxins were preferentially released to the apical side. Unconjugated AOH but not AME aglycone reached the basolateral chamber. The apparent permeability coefficients (Papp values) were calculated for the aglycones as well as total mycotoxin-associated compounds using an initial apical concentration of 20 µmol/l AOH or AME. Based on these Papp values, AOH must be expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycone and as glucuronide and sulfate. In contrast, intestinal absorption of AME appears to be poor and sluggish, with no AME agylcone and only AME conjugates reaching the portal blood.

Oligodeoxynucleotides induce brain inflammation in rats when infused intracerebroventricularly.

Antisense oligodeoxynucleotides (ODNs) can inhibit gene expression in a specific manner. However, several studies described problems with cerebral ODN application. Here, we investigated the immune effects (interleukin-6 (IL-6) release, cell invasion into cerebrospinal fluid (CSF) and brain parenchyma) of 'non-sense' randomized ODNs with different counterions (NH(4)(+), Na(+)) and modifications (with or without thioat-backbone) which were administered intracerebroventricularly for 48 h using osmotic mini-pumps in a rat model. All animals receiving ODNs showed increased IL-6 levels in the CSF as well as cell invasion into the CSF and brain parenchyma (P<0.05). However, the use of thioat-backbone and ammonium as the counterion induced the highest IL-6 levels (7210+/-1696 pg/ml, P<0.05) and the highest cell numbers in the CSF (31.6+/-15.5x10(5)/ml, P<0.05) as well as brain parenchyma (268.1+/-143.2 HIS-48+ cells/mm(2), P<0.01; and 31.3+/-10.7 OX-6+cells/mm(2), P<0.05) compared with the other groups.