Biotransformation Functions Research Articles

Making waves: Enhancing pollutant biodegradation via rational engineering of microbial consortia

Biodegradation holds promise as an effective and sustainable process for the removal of synthetic chemical pollutants. Nevertheless, rational engineering of biodegradation for pollutant remediation remains an unfulfilled goal, while chemical pollution of waters and soils continues to advance. Efforts to (i) identify functional bacteria from aquatic and soil microbiomes, (ii) assemble them into biodegrading consortia, and (iii) identify maintenance and performance determinants, are challenged by large number of pollutants and the complexity in the enzymology and ecology of pollutant biodegradation. To overcome these challenges, approaches that leverage knowledge from environmental bio-chem-informatics and metabolic engineering are crucial. Here, we propose a novel high-throughput bio-chem-informatics pipeline, to link chemicals and their predicted biotransformation pathways with potential enzymes and bacterial strains. Our framework systematically selects the most promising candidates for the degradation of chemicals with unknown biotransformation pathways and associated enzymes from the vast array of aquatic and soil bacteria. We substantiated our perspective by validating the pipeline for two chemicals with known or predicted pathways and show that our predicted strains are consistent with strains known to biotransform those chemicals. Such pipelines can be integrated with metabolic network analysis built upon genome-scale models and ecological principles to rationally design fit-for-purpose bacterial communities for augmenting deficient biotransformation functions and study operational and design parameters that influence their structure and function. We believe that research in this direction can pave the way for achieving our long-term goal of enhancing pollutant biodegradation.

Low concentration of indoxacarb interferes with the growth and development of silkworm by damaging the structure of midgut cells

As an important economic insect, Bombyx mori plays an essential role in the development of the agricultural economy. Indoxacarb, a novel sodium channel blocker insecticide, has been widely used for the control of various pests in agriculture and forestry, and its environmental pollution caused by flight control operations has seriously affected the safe production of sericulture in recent years. However, the lethal toxicity and adverse effects of indoxacarb on silkworm remain largely unknown. In this study, the toxicity of indoxacarb on the 5th instar larvae of silkworm was determined, with an LC50 (72 h) of 2.07 mg/L. Short-term exposure (24 h) to a low concentration of indoxacarb (1/2 LC50) showed significantly reduced body weight and survival rate of silkworm larvae. In addition, indoxacarb also led to decreased cocoon weight and cocoon shell weight, but had no significant effects on pupation, adult eclosion, and oviposition. Histopathological and ultrastructural analysis indicated that indoxacarb could severely damage the structure of the midgut epithelial cells, and lead to physiological impairment of the midgut. A total of 3883 differentially expressed genes (DEGs) were identified by midgut transcriptome sequencing and functionally annotated using GO and KEGG. Furthermore, the transcription level and enzyme activity of the detoxification related genes were determined, and our results suggested that esterases (ESTs) might play a major role in metabolism of indoxacarb in the midgut of B. mori. Future studies to examine the detoxification or biotransformation function of candidate genes will greatly enhance our understanding of indoxacarb metabolism in B. mori. The results of this study provide a theoretical basis for elucidating the mechanism of toxic effects of indoxacarb on silkworm by interfering with the normal physiological functions of the midgut.

Current Strategies for Real-Time Enzyme Activation.

Enzyme activation is a powerful means of achieving biotransformation function, aiming to intensify the reaction processes with a higher yield of product in a short time, and can be exploited for diverse applications. However, conventional activation strategies such as genetic engineering and chemical modification are generally irreversible for enzyme activity, and they also have many limitations, including complex processes and unpredictable results. Recently, near-infrared (NIR), alternating magnetic field (AMF), microwave and ultrasound irradiation, as real-time and precise activation strategies for enzyme analysis, can address many limitations due to their deep penetrability, sustainability, low invasiveness, and sustainability and have been applied in many fields, such as biomedical and industrial applications and chemical synthesis. These spatiotemporal and controllable activation strategies can transfer light, electromagnetic, or ultrasound energy to enzymes, leading to favorable conformational changes and improving the thermal stability, stereoselectivity, and kinetics of enzymes. Furthermore, the different mechanisms of activation strategies have determined the type of applicable enzymes and manipulated protocol designs that either immobilize enzymes on nanomaterials responsive to light or magnetic fields or directly influence enzymatic properties. To employ these effects to finely and efficiently activate enzyme activity, the physicochemical features of nanomaterials and parameters, including the frequency and intensity of activation methods, must be optimized. Therefore, this review offers a comprehensive overview related to emerging technologies for achieving real-time enzyme activation and summarizes their characteristics and advanced applications.

The assembly of integrated rat intestinal-hepatocyte cultures.

The jejunum is the segment of the small intestine responsible for several metabolism and biotransformation functions. In this report, we have cultured rat jejunum explants in vitro and integrated them with hepatocyte cultures. We have also investigated the changes in jejunum function at different locations since spatial variations in intestinal functions have been reported previously. We divided the length of the rat jejunum into three distinct regions of approximately 9 cm each. We defined the regions as proximal (adjacent to the duodenum), medial, and distal (adjacent to the ileum). Spatiotemporal variations in functions were observed between these regions within the jejunum. Alkaline phosphatase activity (a marker of enterocyte function), decreased twofold between the proximal and distal regions at 4 hr. Lysozyme activity (a marker of Paneth cell function) increased from the proximal to the distal jejunum by 40% at 24 hr. Mucin‐covered areas, a marker of goblet cell function, increased by twofold between the proximal and distal segments of the jejunum at 24 hr. When hepatocytes were integrated with proximal jejunum explants, statistically higher urea (~2.4‐fold) and mucin (57%) production were observed in the jejunum explants. The integrated intestine‐liver cultures can be used as a platform for future investigations.

Salvianolic Acid B Enhances Hepatic Differentiation of Human Embryonic Stem Cells Through Upregulation of WNT Pathway and Inhibition of Notch Pathway.

Hepatocytes differentiated from human embryonic stem cells (ESCs) could provide a powerful tool for enabling cell-based therapies, studying the mechanisms underlying human liver development and disease, and testing the efficacy and safety of pharmaceuticals. However, currently most in vitro protocols yield hepatocytes with low levels of liver function. In this study, we investigated the potential of Salvianolic acid B (Sal B), an active pharmaceutical compound present in Salvia miltiorrhiza, which has been shown to have an antifibrotic effect in previous studies, to enhance hepatocyte differentiation from human ESCs. After treatment with Sal B, albumin expression and secretion were consistently increased, indicating that Sal B could promote hepatocyte differentiation process. Expression of a large number of important phase 1 and 2 metabolizing enzymes and phase 3 transporters was also increased in treated cells, indicating an enhanced biotransformation function. Our investigations further revealed the activation of Wnt pathway in treated cells, as determined by upregulation of Wnts, which increased amounts of nuclear β-catenin. This increased nuclear β-catenin led in turn to the enhanced expression of T cell factor (TCF) 3 and lymphoid enhancer-binding factor (LEF) 1 which upregulated their downstream targets, cyclin D1 and c-Myc. Notch receptors (Notch1, Notch3), Notch ligand (Jagged2), and Notch receptor targets [hairy and enhancer of split (Hes) 1, 5] were downregulated in treated cells, suggesting that Notch pathway was inhibited. Consistent with the inhibition of Notch pathway, expression of cholangiocyte marker, CK7, was significantly reduced by treatment with Sal B. Numb, a direct transcriptional target of Wnt pathway and a negative regulator of Notch pathway, was upregulated, consistent with activation of Wnt signaling and suppression of Notch signaling. In conclusion, our study demonstrated that Sal B enhanced hepatocyte differentiation from human ESCs through activation of Wnt pathway and inhibition of Notch pathway. Therefore, this study suggests that Sal B can be used as a potential agent to generate more mature hepatocytes for cell-based therapeutics and pharmaceutical studies.

Bioinspired hollow fiber membrane bioreactor using human liver microtissue spheroids

Event Abstract Back to Event Bioinspired hollow fiber membrane bioreactor using human liver microtissue spheroids Loredana De Bartolo1, Haysam M. Ahmed1, 2, Simona Salerno1, Shervin Khakpour1, 2, Sabrina Morelli1 and Lidietta Giorno1 1 National Research Council of Italy - CNR, Institute on Membrane Technology, Italy 2 University of Calabria, Department of Environmental and Chemical Engineering, Italy Introduction: The development of bioartificial devices that are able to favour the liver reconstruction and to modulate cell behaviour is an important challenge in liver tissue engineering. As strategy we realized liver microtissue spheroids in a crossed hollow fiber membrane bioreactor, which was developed for the in vitro long-term perfusion. The bioreactor consists of two bundles of hollow fiber (HF) polyethersulfone (PES) membranes cross-assembled in alternating manner. One bundle of HF membranes provides cells nutrients and metabolites whereas the other HF bundle removes catabolites from cell compartment mimicking in this way the in vivo arterious and venous blood vessels. The combination of these two fiber set creates three compartments: two intraluminal compartments of PES HF in which the medium flows and one extraluminal compartment represented by extracapillary network formed by the fibers in which spheroids are cultured. Materials and Methods: The bioreactor consists of two bundles of PES HF membranes cross-assembled in alternating manner and potted with polyurethane adhesive within glass housing. The membranes were previously characterised in order to establish the physico-chemical, structural and transport properties[1]. The oxygenated medium enters to the membrane bioreactor with a flow rate Qf of 1.5 mL/min that was set on the basis of average retention time. Its fluid dynamics were characterized in terms of cumulative residence time distribution (RTD). Spheroids of primary human hepatocytes were realized in agarose mold and then cultured in the extralumen compartment of the bioreactor. The oxygen uptake, urea synthesis and albumin production rates as well as biotransformation functions were evaluated with time by using specific assays[2]. Numerical analysis of the mass transfer model for oxygen, urea and albumin concentration was performed using COMSOL Multiphysics. Results and Discussion: The geometry of the bioreactor ensured optimal perfusion conditions, which plaid a crucial role together with the surface and transport properties of the HF membranes in guiding the self-assembling of liver microtissue spheroids. Indeed, adjacent spheroids fused in the extracapillary space forming liver microtissues (Fig. 1). The liver microtissue spheroids expressed liver specific functions in terms of urea synthesis, albumin production and diazepam biotransformation at high levels up to 2 weeks. The liver specific functions of the bioreactor were significantly higher with respect to those of the batch system. Furthermore, the bioreactor provided an adequate oxygenation of the liver microtissue spheroids maintaining oxygen concentration levels well above the in vivo liver periportal zone concentration. Conclusion: The bioreactor thanks to its fluid dynamics and mass transfer properties of the fibers allowed to promote the formation liver microtissue spheroids with high specific functionality.

Detection of changes in genes related to antioxidant and biotransformation function in broiler birds fed aflatoxin by real time PCR

Current findings suggest that the AFB1 failed to influence hepatic gene expression of SOD, CAT, EH, GPx and GST-α but increased the gene expression of the two biotransformation genes CYP1A1, and CYP2H1 at the levels used. Results show that changes in genes related to antioxidant and biotransformation function could be detected by real time PCR. Further work to study the expression of antioxidant and biotransformation genes with higher levels of aflatoxin along with beneficial effects of phytochemicals in amolerating the same in broiler birds are under progress.

Biofabrication of Layered Membrane Systems by Using Human Hepatocytes and Endothelial Cells: A Comparative Study

Strategies to overcome in vitro dedifferentiation of hepatocytes preserving key features of the liver phenotype in vitro constitute a major goal in medical applications. The realization of biohybrid membrane systems, utilizing primary human hepatocytes in co-culture with non-parenchymal cells and polymeric membranes with suitable properties, offered interesting opportunities for the design of bioartificial liver. This study was aimed to investigate the morphological and functional behavior of primary human hepatocytes with endothelial cells (ECs) in two different layered co-culture membrane systems, which were realized by using a synthetic polymeric membrane and two different sequential cell seeding methods. We evaluated the heterotypic cell-cell interactions in the different layered membrane systems in order to understand if the variation of the heterotypic interface between hepatocytes and ECs and of cell type interacting with membrane may influence the performance of functional biofabricated hepatocellular constructs. Overall results demonstrated a strong influence of the heterotypic interactions on the performance of the tissue constructs in terms of albumin secretion, urea synthesis and drug biotransformation functions. Keywords: Biohybrid membrane systems, endothelial cells, hepatocytes, hepatocellular constructs, heterotypic interactions, membranes.

Transcriptome sequencing and microarray development for the woodrat (Neotoma spp.): custom genetic tools for exploring herbivore ecology

Massively parallel sequencing has enabled the creation of novel, in-depth genetic tools for nonmodel, ecologically important organisms. We present the de novo transcriptome sequencing, analysis and microarray development for a vertebrate herbivore, the woodrat (Neotoma spp.). This genus is of ecological and evolutionary interest, especially with respect to ingestion and hepatic metabolism of potentially toxic plant secondary compounds. We generated a liver transcriptome of the desert woodrat (Neotoma lepida) using the Roche 454 platform. The assembled contigs were well annotated using rodent references (99.7% annotation), and biotransformation function was reflected in the gene ontology. The transcriptome was used to develop a custom microarray (eArray, Agilent). We tested the microarray with three experiments: one across species with similar habitat (thus, dietary) niches, one across species with different habitat niches and one across populations within a species. The resulting one-colour arrays had high technical and biological quality. Probes designed from the woodrat transcriptome performed significantly better than functionally similar probes from the Norway rat (Rattus norvegicus). There were a multitude of expression differences across the woodrat treatments, many of which related to biotransformation processes and activities. The pattern and function of the differences indicate shared ecological pressures, and not merely phylogenetic distance, play an important role in shaping gene expression profiles of woodrat species and populations. The quality and functionality of the woodrat transcriptome and custom microarray suggest these tools will be valuable for expanding the scope of herbivore biology, as well as the exploration of conceptual topics in ecology.

Different Effects of Plasma from Patients with Acute on Chronic Liver Failure on the Proliferation and Biotransformation Function of C3A Cells

Abstract Objective To investigate the effects of plasma from patients with acute on chronic liver failure on the proliferation and biotransformation function of C3A cells in vitro, and provide experimental data for C3A cells to be efficiently used in the bioartificial liver system. Methods C3A cells were incubated in 100% normal human plasma (NHP) and 100% abnormal plasma (AP) from patients with acute on chronic liver failure. Growth morphology of the two groups were observed under inverted microscope and scanning electron microscope. The method of methyl thiazolyl tetrazolium (MTT) was conducted for the proliferation activities of C3A cells. The cellular apoptosis rates were assessed by the flow cytometer. The biotransformation function of cells was evaluated through diazepam metabolic amount assay. The concentrations of epithelial growth factor (EGF), transforming growth factor-α (TGF-α) and interleukin-1 (IL-1) were detected in plasma of the two groups. Results A: The proliferation activities of C3A cells incubated in 100% AP for 24, 48, 72, 96 and 120 hours were significantly higher than that in 100% NHP (P < 0.01). B: Observation under the inverted microscope indicated that the cells in 100% AP were growing faster than those in 100% NHP after cells attached to the plastic at 24 and 48 hours. The same phenomena was observed under the scanning electronic microscope. C: The C3A cells cultured in both groups of plasma showed the same apoptosis rate at 48 hours and there was no statistical difference. D: The diazepam metabolic value of C3A cells incubated in 100% AP for 24, 72 and 120 hours were lower than that in 100% NHP and were statistically different (P < 0.01). E: The concentrations of TGF-α, EGF and IL-1 in AP were significantly higher than that in NHP (P < 0.01). Conclusions Compared with normal human plasma, the plasma from patients with acute on chronic liver failure has more obvious effect to facilitate the proliferation of C3A cells, but also decreases partial biotransformation function of C3A cells.

Hemoglobin Regulates the Metabolic, Synthetic, Detoxification, and Biotransformation Functions of Hepatoma Cells Cultured in a Hollow Fiber Bioreactor

Hepatic hollow fiber (HF) bioreactors constitute one type of extracorporeal bioartificial liver assist device (BLAD). Ideally, cultured hepatocytes in a BLAD should closely mimic the in vivo oxygenation environment of the liver sinusoid to yield a device with optimal performance. However, most BLADs, including hepatic HF bioreactors, suffer from O2 limited transport toward cultured hepatocytes, which reduces their performance. We hypothesize that supplementation of hemoglobin-based O2 carriers into the circulating cell culture medium of hepatic HF bioreactors is a feasible and effective strategy to improve bioreactor oxygenation and performance. We examined the effect of bovine hemoglobin (BvHb) supplementation (15g/L) in the circulating cell culture medium of hepatic HF bioreactors on hepatocyte proliferation, metabolism, and varied liver functions, including biosynthesis, detoxification, and biotransformation. It was observed that BvHb supplementation supported the maintenance of a higher cell mass in the extracapillary space, improved hepatocyte metabolic efficiency (i.e., hepatocytes consumed much less glucose), improved hepatocyte capacity for drug metabolism, and conserved both albumin synthesis and ammonia detoxification functions compared to controls (no BvHb supplementation) under the same experimental conditions.

Oxidative enzymes in tissues of the catfish (Clarias gariepinus) exposed to varying levels of cadmium

The activities of some oxidative enzymes (aldehyde oxidase, AO, EC 1.2.3.1; xanthine oxidase, XO, EC 1.17.3.2; sulphite oxidase, SO, EC 1.8.3.1; and monoamine oxidase, MO, EC 1.4.3.4) were investigated in the liver, gill, kidney, brain and muscle of a variety of African catfish (Clarias gariepinus) after 7 and 21 days of exposure to 0.1, 0.2 and 0.4 ppm cadmium (Cd) in ambient water. MO, SO and AO activities were significantly (p < 0.05) decreased in a dose-dependent manner in the liver, kidney and gill in the 7-day Cd-exposed fish when compared to control. Conversely, brain MO, AO and SO activities were significantly (p < 0.05) elevated in a dose-dependent manner in the same fishes relative to control. As in the 7-day Cd-exposed fish, the activities of MO, AO and SO were significantly (p < 0.05) decreased in a dose-dependent manner in the gill, kidney and liver of catfish exposed for 21 days. Same is true for XO in these organs after both duration of exposure, except in the liver where no significant change was observed relative to control. The inhibition of these oxidative enzymes in the liver, gill and kidney of catfish may interfere with their biotransformation function, and ultimately the survival of the fish may be compromised.

5-ketoreductase from Streptomyces bingchengensis: overexpression and preliminary characterization

To elucidate the biotransformation from 5-oxomilbemycins A(3) and A(4) to milbemycins A(3) and A(4) in Streptomyces bingchengensis, the C5-ketoreductase gene (milF) was cloned using PCR with the specific primer designed from homologous nucleotide sequences. The C5-ketoreductase (MilF) was heterologously expressed in E. coli BL21 (DE3) as a His-tagged fusion protein. The characterization and biotransformation function of purified MilF was verified by in vitro enzyme assay. MilF is an NADPH-dependent reductase. The biotransformation products, analyzed by LC-APCI/MS, were identified as milbemycin A(3) and milbemycin A(4). MilF is thus present in Streptomyces bingchengensis and can transform 5-oxomilbemycins A(3) and A(4) to milbemycins A(3) and A(4). These findings are significant for understanding the biosynthetic pathway of milbemycins in Streptomyces bingchengensis and pave the way to obtain a producer strain of 5-oxomilbemycins directly by targeted milF disruption.

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg 2+) and organic mercury (CH 3Hg +): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg 2+) and organic (CH 3Hg +) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC 50) = 250 nM and k inact = 1.4 × 10 4 M −1 s −1 for Hg 2+ and IC 50 = 1.4 μM and k inact = 2 × 10 2 M −1 s −1 for CH 3Hg +). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC 50 = 3 and 20 μM for Hg 2+ and CH 3Hg +, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.

Genetic association of phase I and phase II detoxification genes with recurrent miscarriages among North Indian women

Allelic variants of the detoxification genes that have impaired biotransformation functions may increase susceptibility to reproductive toxicity leading to endometriosis, recurrent miscarriage (RM) or poor pregnancy outcome. In the present study, we have investigated CYP1A1, CYP2D6, GSTT1, GSTP1 and GSTM1, which are involved in the phase I and phase II detoxification systems, in relation to their role in the etiology of unexplained RMs. In a case-control study, we have investigated 200 females with RM and 300 age and ethnically matched healthy controls with successful reproductive history from North India. The frequencies of phase I wild-type genotypes of CYP1A1 and CYP2D6 in RM cases were 0.56 and 0.60, whereas in controls these were 0.68 and 0.65, respectively (both P < 0.05). The GSTM1 null-genotype frequencies were 0.66 and 0.84 among RM cases and controls, respectively, the GSTT1 null-genotype frequencies were 0.52 and 0.45 (P < 0.005) and the GSTP1 variant allele frequencies were 0.23 and 0.20, respectively. In conclusion, we observed significant protective effects of phase I wild-type genotypes and association of the GSTT1 null genotype with RM. Through combined analyses we have highlighted the importance of the balance of phase I/phase II detoxification systems, in the etiology of RM.

Novel bioactive polymeric membranes to elicit specific human hepatocyte responses

The aim of the paper was the design and development of new bioactive polymeric membranes to be used for the in vitro reconstruction of liver tissue-model [1–2]. Significant efforts have also been made to develop new polymeric materials able to favour the liver reconstruction and to modulate liver cell behaviour. We developed membranes from a polymeric blend of modified polyether-etherketone (PEEK-WC) and polyurethane (PU) by inverse phase technique by using the direct immersionprecipitation method (PEEK-WC-PU) [3]. The morphological and physico-chemical properties of the PEEK-WC-PU membranes were established before their use in the cell culture system. Preliminarily a screening of this new material was performed investigating the specific liver functions in terms of albumin and urea synthesis using primary human hepatocytes (Cambrex Bio Science) cultured on PEEK-WC-PU membranes in comparison with collagen film. The biotransformation functions of human liver cells were evaluated using diclofenac (DIC), a nonsteroidal anti-inflammatory drug that is widely used in painful and inflammatory rheumatic and nonrheumatic conditions. Then the performance of the novel PEEK-WC-PU membranes was validated by using the biohybrid system constituted by human hepatocytes and PEEK-WC-PU membrane in a oxygen permeable membrane bioreactor. Liver specific functions in terms of urea synthesis and total protein secretion was evaluated in the time. Drug biotransformation was also assessed using Diazepam, an anti anxiety agent (benzodiazepines), as model drug. *Corresponding author.

Galactose Derivative Immobilized Glow Discharge Processed Polyethersulfone Membranes Maintain the Liver Cell Metabolic Activity

New strategies aimed to surface modification of polymeric membranes are crucial to optimise cell-biomaterial interactions in vivo and in vitro biohybrid systems. In this paper, we investigated the surface modification of Polyethersulfone (PES) membranes by plasma polymerisation of acrylic acid monomers (PES-pdAA) and by immobilization of galactonic acid through a hydrophilic "spacer arm" molecule (PES-pdAA-SA-GAL). The modification steps were characterised by high resolution X-ray photoelectron spectroscopy. The performance of modified and unmodified membranes was evaluated by assessing the expression of liver specific biotransformation functions of pig and human hepatocytes. Human liver cells cultured on PES-pdAA-SA-GAL membranes displayed an enhanced albumin production, urea synthesis and protein secretion for 24 days of culture. The immobilisation of galactose derivative units on the membrane allowed specific interactions with hepatocytes biomimicking the cellular microenvironment and produced an improvement of the long-term maintenance and differentiation of human hepatocytes.

Long-term maintenance of human hepatocytes in oxygen-permeable membrane bioreactor

An oxygen-permeable membrane bioreactor utilizing human hepatocytes has been tested in this study. In the bioreactor, human hepatocytes were cultured between flat-sheet gas-permeable polymeric membranes, which ensure the diffusion of O(2) and CO(2) providing a support for cell anchorage and growth and permit the online observation of the cells with an inverse microscope. This bioreactor allows a direct oxygenation of cells adhered on membranes and of the medium overlaying cells simulating in vivo sinusoidal organization. Human hepatocytes were cultured in the presence of some therapeutic molecules to assess the temporal liver-specific functions of the cells. Interleukin 6 (IL-6), which is a multifactorial proinflammatory cytokine involved in a variety of host defences and pathological processes, and diclofenac, an arylacetic non-steroidal anti-inflammatory drug, were used as therapeutic molecules. The aim of this study was to evaluate the in vitro performance of the small oxygen-permeable membrane bioreactor in the long-term maintenance and differentiation of human hepatocytes under in-vivo-like conditions. The fluid dynamics of the bioreactor were characterized before using it for human cell culture. The functional response to a step challenge in the medium of IL-6 (120 pg/ml), diclofenac (80 microm) and IL-6 and diclofenac together was investigated. The ability of hepatocytes to perform liver-specific functions in terms of urea and albumin synthesis, as well as secretion of total proteins, was maintained for 32 days. Also, the diclofenac biotransformation functions were sustained as the formation of the metabolites 4'-OH-diclofenac and 5-OH-diclofenac lactam demonstrated. This study attested the feasibility of the membrane bioreactor as an in vitro simple model system that allows human hepatocytes to be maintained in a differentiated state similar to that in vivo.

Polyethersulfone membrane biohybrid system using pig hepatocytes: Effect of diclofenac on cell biotransformation and synthetic functions

A biohybrid system constituted of primary pig hepatocytes and microporous synthetic membranes was used to investigate the diclofenac biotransformation and its long-term effect on hepatocyte specific functions. The maintenance of drug biotransformation functions and the effect of diclofenac continuous administration on the viability and metabolic functions of hepatocytes have been explored. Pig hepatocytes were isolated from slaughterhouse organs and were cultured on polyethersulfone membranes and on collagen. The ability of cells to eliminate diclofenac and to synthesise metabolites was evaluated by an HPLC analysis. Urea and albumin productions were established as indicators of differentiated hepatocyte functions. Pig hepatocytes eliminated diclofenac and produced two main metabolites: 4′-hydroxydiclofenac (4′-OHdic) and N,5-dihydroxydiclofenac ( N,5-(OH) 2dic). Cells continuously exposed to subtoxic drug concentrations maintained their biotransformation functions until 10 days of culture. The rate of diclofenac elimination increased according to a saturation kinetics. Diclofenac showed a cytotoxic effect at a concentration higher than 100 μM as confirmed by analysis of DNA fragmentation. The urea and albumin synthesis as well as the total protein production were also affected by diclofenac concentrations. An important concentration-dependent effect of diclofenac on protein synthesis was observed. The results give information concerning the effect of diclofenac on specific functions of pig hepatocytes continuously exposed to the drug and on the long-term ability of cells to metabolise diclofenac in a membrane biohybrid system.

A Microscale In Vitro Physiological Model of the Liver: Predictive Screens for Drug Metabolism and Enzyme Induction

In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery -- as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.