Evidence of renal metabolism of ifosfamide to nephrotoxic metabolites

Nephrotoxicity is a limiting factor in the use of ifosfamide in children. Despite the co-administration of uroprotective agents such as sodium 2-mercaptoethanesulfonate (mesna), ifosfamide chemotherapy is associated with nephropathy characterized by glomerular toxicity and Fanconi syndrome in many children treated with this drug. This is in distinction to cyclophosphamide, an analogue which differs solely by the position of a chloroethyl group, and which is not associated with nephrotoxicity. We hypothesized that ifosfamide is metabolized by cytochrome P450 (CYP) enzymes located in the renal tubular cell to the toxic metabolite chloroacetaldehyde; and, that the higher production of chloroacetaldehyde from ifosfamide than from cyclophosphamide explains the clinical differences in nephrotoxicity. We found that in both pig renal cortical microsomes and whole human kidney microsomes incubated with 1 mM ifosfamide for 3 hr, 2 and 3 dechloroethylifosfamide (DCEI) were produced. Our study provides evidence that porcine and human kidney microsomes are capable of biotransforming ifosfamide to DCEI metabolites that are produced in equimolar amounts with chloroacetaldehyde, indicating that chloroacetaldehyde is locally produced by renal cells as a possible mechanism for nephrotoxicity.     

Toxicity of ifosfamide and its metabolite chloroacetaldehyde in cultured renal tubule cells

Summary Renal injury is a common side effect of the chemotherapeutic agent ifosamide. Current evidence suggests that the ifosfamide metabolite chloroacetaldehyde may contribute to this nephrotoxicity. The present study examined the effects of ifosfamide and chloroacetaldehyde on rabbit proximal renal tubule cells in primary culture. The ability of the uroprotectant medication sodium 2-mercaptoethanesulfonate (mesna) to prevent chloroacetaldehyde-induced renal cell injury was also assessed. Chloroacetaldehyde (12.5–150 μM) produced dose-dependent declines in neutral red dye uptake, ATP levels, glutathione content, and cell growth. Coadministration of mesna prevented chloroacetaldehyde toxicity while pretreatment of cells with the glutathione-depleting agent buthionine sulfoximine enhanced the toxicity of chloroacetaldehyde. Ifosfamide (1000–10 000 μM) toxicity was detected only at concentrations of 4000 μM or greater. Analysis of media collected from ifosfamide-treated cell cultures revealed the presence of several ifosfamide metabolites, demonstrating that renal proximal tubule cells are capable of biotransforming this chemotherapeutic agent. This primary renal cell culture system should prove useful in studying the cause and prevention of ifosfamide nephrotoxicity.     

A tubule cell model for ifosfamide nephrotoxicity

Mechanisms leading to ifosfamide (IF)-induced renal damage have not been fully elucidated. Recent work suggests that localized renal tubular metabolism of IF and the production of the nephrotoxic chloroacetaldehyde may lead to nephrotoxicity. Presently no pharmacological method to reduce IF nephrotoxicity has been identified. The objectives of this study were to establish a tubule cell model for IF nephrotoxicity, to verify whether renal proximal tubular cells have the necessary cytochrome P450 (CYP) enzymes to oxidize IF, and whether they can metabolize IF to chloroacetaldehyde. CYP3A, and 2B mRNA and protein were identified in LLCPK-1 cells. The cells metabolized the R- and S-IF enantiomers to their respective 2- and 3-dechloroethylifosfamide metabolites, by-products of chloroacetaldehyde formation. Metabolite production was both time and concentration-dependent. IF did not affect cell viability. In contrast, glutathione-depleted cells showed time and dose-dependent damage. The presence of the relevant CYP enzymes in renal tubular cells along with their ability to metabolize IF to its 2- and 3-dechloroethylifosfamide metabolites suggests that nephrotoxic damage may result from the localized production of chloroacetaldehyde. Glutathione is a major defence mechanism against IF toxicity, thus pharmacological methods for replenishing intracellular glutathione may be effective in modulating IF-induced nephrotoxicity.     

Gas‐permeable membranes and co‐culture with fibroblasts enable high‐density hepatocyte culture as multilayered liver tissues

To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three‐dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell–cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate‐based format, enabling high density hepatocyte culture as a stable 3D‐multilayer. Multilayered co‐cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen‐conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co‐cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate‐based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co‐cultures were maintained for at least 1 week; the so‐cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate‐based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.     

Novel approach for the prediction of cell densities and viability in standardized translucent cell culture biochips with near infrared spectroscopy

Near infrared spectroscopy is a rapid and nondestructive method for compositional analysis of biological material. The technology is widely used within bioreactors and possesses potential as a standardized method for quality control in miniaturized microfluidic cell culture systems. Here, we established a method for quantification of cell density and viability of adherent HepaRG cells cultured in a translucent, miniaturized cell culture biochip. The newly developed statistical models for interpretation of near infrared spectroscopy from biochips are the basis for a novel method of fast, continuous, and contact‐free analysis of cell viability and real‐time monitoring of cell growth. The technique thus paves the way for a robust and reliable high‐throughput analysis of biochip‐embedded cell cultures.     

Transcriptomic analysis of the effect of ifosfamide on MDCK cells cultivated in microfluidic biochips

We investigated the behavior of renal cells cultivated in microfluidic biochips when exposed to 50 μM of ifosfamide, an antineoplastic drug treatment. The microarray analysis revealed that ifosfamide had any effect in Petri conditions. The microfluidic biochips induced an early inflammatory response in the MDCK in the untreated cells. This was attributed to cells adapting to the dynamics and micro environment created by the biochips. This led to modulations in the mitochondria dysfunction pathway, the Nrf-2 and oxidative stress pathways and some related cancer genes. When exposed to 50 μM of ifosfamide, we detected a modulation of the pathways related to the cancer and inflammation in the MDCK cultivated in the biochips via modulation of the ATM, p53, MAP Kinase, Nrf-2 and NFKB signaling. In addition, the genes identified and related proteins affected by the ifosfamide treatment in the biochips such as TXNRD1, HSP40 (DNAJB4 and DNAJB9), HSP70 (HSPA9), p21 (CDKN1A), TP53, IKBalpha (NFKBIA) are reported to be the molecular targets in cancer therapy. We also found that the integrin pathway was perturbed with the ifosfamide treatment. Finally, the MYC proto-oncogene appeared to be a potential bridge between the integrin signaling and the anti-inflammatory response.     

Stepwise assembly of micropatterned co‐cultures using photoresponsive culture surfaces and its application to hepatic tissue arrays

Cell micropatterning, a method to place cells at arbitrary regions, is becoming an essential tool to conduct cell biology and tissue engineering. Conventional cell patterning techniques usually allow only single patterning with single cell type on the same culture surface. However, biomedical research today requires even sophisticated fabrication methods that require spatiotemporal control of multiple cell arrangements. Here we introduce in situ cell micropatterning system which enables stepwise cell patterning using a photoresponsive cell culture surface (PRCS) whose cell adhesiveness could be altered by the UV irradiation. To demonstrate an application to tissue engineering, a liver‐mimic tissue array was fabricated and liver‐specific gene expressions were quantified with real time PCR. Patterned co‐culture systems composed of HepG2 spheroids with Balb/3T3 were fabricated, and the optimum spheroid diameter, which yielded the highest cellular functions, was determined to be 150 µm. After 20 days of patterned co‐culture of HepG2 spheroids and Balb/3T3, CYP3A4 expression increased 50‐fold higher than conventionally cultured HepG2; CYP3A4 expression was 20% higher than randomly co‐cultured HepG2 and Balb/3T3. Thus the combination of PRCS and the photomask‐free irradiation apparatus showed the versatility of experimental setups and proved to be a powerful tool for biomedical studies. Biotechnol. Bioeng. 2009;103: 552–561. © 2009 Wiley Periodicals, Inc.     

Madin–Darby canine kidney cells are increased in aerobic glycolysis when cultured on flat and stiff collagen‐coated surfaces rather than in physiological 3‐D cultures

We investigate the influence of the dimensionality and the biochemistry of the culture system on the cellular functionality by analyzing the protein expression levels in Madin–Darby canine kidney (MDCK) cells grown in 3‐D and 2‐D substrates. We cultured MDCK cells on a hard and flat 2‐D uncoated plastic surface, on a 2‐D collagen‐coated plastic surface and in 3‐D collagen gel and employed 2‐D gel electrophoresis, MALDI‐TOF‐MS, and LC‐MS/MS analysis to identify the differentially regulated proteins. We found significant differences in the expression of antioxidant proteins, actin‐binding proteins, glycolytic enzymes, and heat‐shock proteins/chaperons among the three types of cultures. While MDCK cells cultured in 3‐D collagen up‐regulate antioxidant proteins and proteins involved in the dynamic remodeling of the actin cytoskeleton, 2‐D collagen‐coated plastic surfaces induce the up‐regulation of glycolytic enzymes. Our data shows that the culture conditions have profound effects on the physiology of the cell. Culture in 3‐D collagen induces a differentiated polarized phenotype. In contrast, collagen‐coated 2‐D substrates favor a tumor‐like phenotype with increased glycolysis. Thus, the suitability of 2‐D cultures to study the physiological behavior of cells, especially in drug discovery, bioprocessing, and toxicology, should be carefully reconsidered.     

3D spheroid cultures improve the metabolic gene expression profiles of HepaRG cells

3D (three-dimensional) cultures are considered to be an effective method for toxicological studies; however, little evidence has been reported whether 3D cultures have an impact on hepatocellular physiology regarding lipid or glucose metabolism. In the present study, we conducted physiological characterization of hepatoma cell lines HepG2 and HepaRG cells cultured in 3D conditions using a hanging drop method to verify the effect of culture environment on cellular responses. Apo (Apolipoprotein)B as well as albumin secretion was augmented by 3D cultures. Expression of genes related to not only drug, but also glucose and lipid metabolism were significantly enhanced in 3D cultured HepaRG spheroids. Furthermore, mRNA levels of CYP (cytochrome P450) enzymes following exposure to corresponding inducers increased under the 3D condition. These data suggest that this simple 3D culture system without any special biomaterials can improve liver-specific characteristics including lipid metabolism. Considering that the system enables high-throughput assay, it may become a powerful tool for compound screening concerning hepatocellular responses in order to identify potential drugs.     

Perfusion flow rate substantially contributes to the performance of the HepaRG‐AMC‐bioartificial liver

Bioartificial livers (BALs) are bioreactors containing liver cells that provide extracorporeal liver support to liver‐failure patients. Theoretically, the plasma perfusion flow rate through a BAL is an important determinant of its functionality. Low flow rates can limit functionality due to limited substrate availability, and high flow rates can induce cell damage. This hypothesis was tested by perfusing the AMC‐BAL loaded with the liver cell line HepaRG at four different medium flow rates (0.3, 1.5, 5, and 10 mL/min). Hepatic functions ammonia elimination, urea production, lactate consumption, and 6β‐hydroxylation of testosterone showed 2–20‐fold higher rates at 5 mL/min compared to 0.3 mL/min, while cell damage remained stable. However, at 10 mL/min cell damage was twofold higher, and maximal hepatic functionality was not changed, except for an increase in lactate elimination. On the other hand, only a low flow rate of 0.3 mL/min allowed for an accurate measurement of the ammonia and lactate mass balance across the bioreactor, which is useful for monitoring the BAL's condition during treatment. These results show that (1) the functionality of a BAL highly depends on the perfusion rate; (2) there is a universal optimal flow rate based on various function and cell damage parameters (5 mL/min for HepaRG‐BAL); and (3) in the current set‐up the mass balance of substrate, metabolite, or cell damage markers between in‐and out‐flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate (0.3 mL/min for HepaRG‐BAL). Biotechnol. Bioeng. 2012; 109: 3182–3188. © 2012 Wiley Periodicals, Inc.     

Enantioselective metabolism of ifosfamide by the kidney

Ifosfamide (IF), a potent chemotherapeutic agent for solid tumors, is known to cause high rates of nephrotoxicity, which is most likely due to the renal production of the metabolite chloroacetaldehyde. Enantioselective oxidation of IF has been shown in the liver but has never been reported in the kidney. Using porcine and human kidney samples, as well as the renal porcine cell line LLCPK-1, we document enantioselective metabolism of IF with prevalent production of the N-dechloroethylifosfamide (DCEIF) metabolites from the (S)-IF enantiomer compared to the amount of N-DCEIF metabolites produced from the (R)-IF enantiomers. Since IF enantiomers appear to be equally effective in chemotherapy, these results suggest that replacing the clinically standard racemic mixture of IF with (R)-IF may decrease renal metabolism of the drug and hence may decrease nephrotoxicity.     

TAT‐mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression

Although some studies have shown that the cell penetrating peptide (CPP) TAT can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivo environment. The mechanisms underlying TAT‐mediated transduction have been investigated in cell lines, but not in primary brain cells. In this study we demonstrate that transduction of a green fluorescent protein (GFP)‐TAT fusion protein is dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes. GFP‐TAT transduced PC12 cells and did so with even higher efficiency following NGF differentiation. In cultures of primary brain cells, TAT significantly enhanced GFP delivery into astrocytes grown under different conditions: (1) monocultures grown in serum‐containing medium; (2) monocultures grown in serum‐free medium; (3) cocultures with neurons in serum‐free medium. The efficiency of GFP‐TAT transduction was significantly higher in the monocultures than in the cocultures. The GFP‐TAT construct did not significantly enter neurons. Experimental modulation of GAG content correlated with alterations in TAT transduction in PC12 cells and astrocyte monocultures grown in the presence of serum. In addition, this correlation was predictive of TAT‐mediated transduction in astrocyte monocultures grown in serum free medium and in coculture. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT‐mediated transduction efficiency, extending previous results from cell lines to primary cells. These results highlight the cell‐type and phenotype‐dependence of TAT‐mediated transduction, and underscore the necessity of controlling the phenotype of the target cell in future protein engineering efforts aimed at creating more efficacious CPPs. Biotechnol. Bioeng. 2009; 104: 10–19 © 2009 Wiley Periodicals, Inc.     

犬肾细胞MDCK无血清贴壁及单细胞悬浮培养

旨在挖掘用于鉴定金黄色葡萄球菌的高特异性靶点及其PCR检测引物。采用C++语言编程,以金黄色葡萄球菌Staphylococcus aureus MRSA 252基因组编码序列为对象,对2 656个可编码区进行分析,获得特异性靶点序列,并设计PCR扩增引物。对包括葡萄球菌属11个种及其他细菌属在内的共计137株细菌验证引物特异性,筛选获得9个DNA序列,并设计了4对引物。经验证2对引物的特异性较好,其中引物SA3的基因组DNA检测限为13.7 fg/μL,菌体检测限为9.25×102 CFU/mL。结果验证     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?     

Direct cell–cell contact between periodontal ligament fibroblasts and osteoclast precursors synergistically increases the expression of genes related to osteoclastogenesis

The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell–cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell–cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co‐culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule‐1 (ICAM‐1) and of osteoclastogenesis‐related genes (RANKL, RANK, TNF‐α, and IL‐1β) was highly up‐regulated in the co‐cultures compared to mono‐cultures and the 5–10‐fold up‐regulation reflected a synergistic increase due to direct cell–cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)₂VitD₃ and dexamethasone. In case of indirect cell–cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast‐like cells that were formed in co‐culture with periodontal ligament fibroblasts was significantly augmented compared to mono‐cultures. Our data indicate that cell–cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts. J. Cell. Physiol. 222: 565–573, 2010. © 2009 Wiley‐Liss, Inc.     

Perfusion enhanced polydimethylsiloxane based scaffold cell culturing system for multi‐well drug screening platform

Conventional two‐dimensional cultures in monolayer and sandwich configuration have been used as a model for in vitro drug testing. However, these culture configurations do not present the actual in vivo liver cytoarchitecture for the hepatocytes cultures and thus they may compromise the cells liver‐specific functions and their cuboidal morphology over longer term culture. In this study, we present a three‐dimensional polydimethylsiloxane (PDMS) scaffold with interconnected spherical macropores for the culturing of rat liver cells (hepatocytes). The scaffolds were integrated into our perfusion enhanced bioreactor to improve the nutrients and gas supply for cell cultures. The liver‐specific functions of the cell culture were assessed by their albumin and urea production, and the changes in the cell morphology were tracked by immunofluorescence staining over 9 days of culture period. N‐Acetyl‐Para‐Amino‐Phenol (acetaminophen) was used as drug model to investigate the response of cells to drug in our scaffold‐bioreactor system. Our experimental results revealed that the perfusion enhanced PDMS‐based scaffold system provides a more conducive microenvironment with better cell‐to‐cell contacts among the hepatocytes that maintains the culture specific enzymatic functions and their cuboidal morphology during the culturing period. The numerical simulation results further showed improved oxygen distribution within the culturing chamber with the scaffold providing an additional function of shielding the cell cultures from the potentially detrimental fluid induced shear stresses. In conclusion, this study could serve a crucial role as a platform for future preclinical hepatotoxicity testing. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:418–428, 2014     

Renal proximal tubular cells in suspension or in primary culture as in vitro models to study nephrotoxicity

The kidney forms a frequent target for xenobiotic toxicity. The complex biochemical mechanisms underlying nephrotoxicity are best studied in vitro provided that reliable and relevant in vitro models are available. Since most nephrotoxicants affect primarily the cells of the proximal tubules (PTC), much effort has been directed towards the development of in vitro models of PTC. This review focuses on the preparation of PTC and the use of these cells. Discussed are important criteria such as the viability (survival time) of the cells and the parameters to assess toxicity. Recent studies have shown that isolated PTC in suspension are especially suitable for studies on the biochemical mechanisms of 'acute' nephrotoxicity, whereas PTC in primary culture may be used to investigate mechanisms of nephrotoxic damage at very low concentrations, upon prolonged exposure. PTC cultured on porous filter membranes provide new possibilities to study toxicity in relation to cell and transport polarity. Primary cell cultures of human PTC have been set up. Although a further characterization of these systems is needed, recent data indicate their usefulness.     

Physiological and proteomic analyses of Fe(III)‐reducing co‐cultures of Desulfotomaculum reducens MI‐1 and Geobacter sulfurreducens PCA

We established Fe(III)‐reducing co‐cultures of two species of metal‐reducing bacteria, the Gram‐positive Desulfotomaculum reducens MI‐1 and the Gram‐negative Geobacter sulfurreducens PCA. Co‐cultures were given pyruvate, a substrate that D. reducens can ferment and use as electron donor for Fe(III) reduction. G. sulfurreducens relied upon products of pyruvate oxidation by D. reducens (acetate, hydrogen) for use as electron donor in the co‐culture. Co‐cultures reduced Fe(III) to Fe(II) robustly, and Fe(II) was consistently detected earlier in co‐cultures than pure cultures. Notably, faster cell growth, and correspondingly faster pyruvate oxidation, was observed by D. reducens in co‐cultures. Global comparative proteomic analysis was performed to observe differential protein abundance during co‐culture vs. pure culture growth. Proteins previously associated with Fe(III) reduction in G. sulfurreducens, namely c‐type cytochromes and type IV pili proteins, were significantly increased in abundance in co‐cultures relative to pure cultures. D. reducens ribosomal proteins were significantly increased in co‐cultures, likely a reflection of faster growth rates observed for D. reducens cells while in co‐culture. Furthermore, we developed multiple reaction monitoring (MRM) assays to quantitate specific biomarker peptides. The assays were validated in pure and co‐cultures, and protein abundance ratios from targeted MRM and global proteomic analysis correlate significantly.