Inter-individual Variability in Activity of the Major Drug Metabolizing Enzymes in Liver Homogenates of 20 Individuals

Braver-Sewradj, Shalenie P. den; Braver, Michiel W. den; Dijk, Marc van; Zhang, Yongjie; Dekker, Stefan J.; Wijaya, Lukas S; Vermeulen, Nico; Richert, Lysiane; Commandeur, Jan N. M.; Vos, J. Chris

doi:10.2174/1389200219666180108160046

Cited by 19 publications

(9 citation statements)

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“…Such new platforms are especially needed in biomedical research, as liver damage is a primary cause for post-marketing withdrawal of new drugs, [41] a situation accentuated by the fact that current animal and cell culture models are insufficient to fully predict human physiology or donor-dependent responses. [42,43] The performance of liver organoids within the selected hydrogel upon bioprinting via VBP was investigated. Given the inherent challenge in replicating the multifaceted biosynthetic functions of native hepatocytes in vitro, we specifically analyzed i) the viability of the printed structures, and ii) the influence of the VBP on organoid microstructure and morphology, as a preliminary step to promote the differentiation and maturation capacity of the printed construct into hepatic-like structures.…”

Section: Resultsmentioning

confidence: 99%

Volumetric Bioprinting of Organoids and Optically Tuned Hydrogels to Build Liver‐Like Metabolic Biofactories

et al. 2022

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Organ‐ and tissue‐level biological functions are intimately linked to microscale cell–cell interactions and to the overarching tissue architecture. Together, biofabrication and organoid technologies offer the unique potential to engineer multi‐scale living constructs, with cellular microenvironments formed by stem cell self‐assembled structures embedded in customizable bioprinted geometries. This study introduces the volumetric bioprinting of complex organoid‐laden constructs, which capture key functions of the human liver. Volumetric bioprinting via optical tomography shapes organoid‐laden gelatin hydrogels into complex centimeter‐scale 3D structures in under 20 s. Optically tuned bioresins enable refractive index matching of specific intracellular structures, countering the disruptive impact of cell‐mediated light scattering on printing resolution. This layerless, nozzle‐free technique poses no harmful mechanical stresses on organoids, resulting in superior viability and morphology preservation post‐printing. Bioprinted organoids undergo hepatocytic differentiation showing albumin synthesis, liver‐specific enzyme activity, and remarkably acquired native‐like polarization. Organoids embedded within low stiffness gelatins (<2 kPa) are bioprinted into mathematically defined lattices with varying degrees of pore network tortuosity, and cultured under perfusion. These structures act as metabolic biofactories in which liver‐specific ammonia detoxification can be enhanced by the architectural profile of the constructs. This technology opens up new possibilities for regenerative medicine and personalized drug testing.

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Section: Resultsmentioning

confidence: 99%

Volumetric Bioprinting of Organoids and Optically Tuned Hydrogels to Build Liver‐Like Metabolic Biofactories

et al. 2022

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“…Currently, primary human hepatocytes (pHHs) are the "gold standard" for in vitro studies on hepatic metabolism, clearance, hepatotoxicity and drug-drug interaction [9]. However, this research is still restricted by pHH scarcity, donor variability and their rapid dedifferentiation in vitro [10][11][12][13][14]. An inflammatory response by endotoxin contamination [15,16] originating from bacterial collagenase preparations, loss of normal cell polarity when dissolving them from liver tissue or down-regulation of liver-specific transcription factors influencing phase I/II protein expression were discussed as possible causes [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies

Schulz

Kammerer

Küpper

2019

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BACKGROUND: Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte ® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte ® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes. OBJECTIVE: For Upcyte ® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte ® hepatocytes representing proliferating primary-like hepatocytes. METHODS: POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method. RESULTS: The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3

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“…The results showed that fecal GUS difference across individuals is significant (4~6 folds). Although microbial GUS activity is highly different across individual rats, this variation did not exceed the individual variations of phase I and II enzymes (e.g., CYPs and UGTs) activities [ 38 , 39 ]. In order to determine precision activity, researchers should pay more attention towards individual variation as it has never been reported in literature.…”

Section: Discussionmentioning

confidence: 99%

Glucuronides Hydrolysis by Intestinal Microbial β-Glucuronidases (GUS) Is Affected by Sampling, Enzyme Preparation, Buffer pH, and Species

et al. 2021

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Glucuronides hydrolysis by intestinal microbial β-Glucuronidases (GUS) is an important procedure for many endogenous and exogenous compounds. The purpose of this study is to determine the impact of experimental conditions on glucuronide hydrolysis by intestinal microbial GUS. Standard probe 4-Nitrophenyl β-D-glucopyranoside (pNPG) and a natural glucuronide wogonoside were used as the model compounds. Feces collection time, buffer conditions, interindividual, and species variations were evaluated by incubating the substrates with enzymes. The relative reaction activity of pNPG, reaction rates, and reaction kinetics for wogonoside were calculated. Fresh feces showed the highest hydrolysis activities. Sonication increased total protein yield during enzyme preparation. The pH of the reaction system increased the activity in 0.69–1.32-fold, 2.9–12.9-fold, and 0.28–1.56-fold for mouse, rat, and human at three different concentrations of wogonoside, respectively. The Vmax for wogonoside hydrolysis was 2.37 ± 0.06, 4.48 ± 0.11, and 5.17 ± 0.16 μmol/min/mg and Km was 6.51 ± 0.71, 3.04 ± 0.34, and 0.34 ± 0.047 μM for mouse, rat, and human, respectively. The inter-individual difference was significant (4–6-fold) using inbred rats as the model animal. Fresh feces should be used to avoid activity loss and sonication should be utilized in enzyme preparation to increase hydrolysis activity. The buffer pH should be appropriate according to the species. Inter-individual and species variations were significant.

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Inter-individual Variability in Activity of the Major Drug Metabolizing Enzymes in Liver Homogenates of 20 Individuals

Cited by 19 publications

References 98 publications

Volumetric Bioprinting of Organoids and Optically Tuned Hydrogels to Build Liver‐Like Metabolic Biofactories

Volumetric Bioprinting of Organoids and Optically Tuned Hydrogels to Build Liver‐Like Metabolic Biofactories

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies

Glucuronides Hydrolysis by Intestinal Microbial β-Glucuronidases (GUS) Is Affected by Sampling, Enzyme Preparation, Buffer pH, and Species

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