Biology subjects

Gicquel, T.

Publications and source records attributed to Gicquel, T..

4 recordsLinked to original sources

bioRxiv2025

Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

Primary cultures of Human Hepatocytes (PHH) are the gold standard to investigate drug-hepatotoxicity in vitro, however, large-scale studies using these primary liver cells are not possible because of the shortage in liver biopsies. HepaRG model is often considered as the closest surrogate to PHH for toxicity studies in vitro. However, differentiated HepaRG cells express very low levels of the cytochrome P450 2D6 (CYP2D6) protein, which is essential for the biotransformation of nearly 25% of drugs on the market. To overcome this limitation, infection of progenitor HepaRG cells were performed using lentiviral particles containing a transgene encoding a single mRNA translated into a polypeptide undergoing proteolytic cleavage via the T2A peptide to produce both CYP2D6 and GFP. Differentiated HepaRG cells transduced with lentivirus stably expressed GFP and catalytically active human CYP2D6 enzyme at levels close to those found in high PHH metabolizers. As expected, CYP2D6 protein was found mostly located in the endoplasmic reticulum. Using the CYP2D6 transgenic HepaRG cells, we showed that tramadol was metabolized in both, N- and O-desmethyl tramadol as observed in human serum in contrast with the production of N-desmethyl tramadol only in parental HepaRG cells via the CYP3A4 catalytic activity. Similarly, after perhexiline (PHX) treatments, higher IC50 were found in CYP2D6 expressing HepaRG cells associated to lower mitochondrial damages compared to those found in parental cells for the same PHX concentrations. Gene profiling between parental and transgenic cells demonstrated that the CYP2D6 expressing HepaRG cells had kept their ability to proliferate and differentiate with low impact on the expression of the hepatocyte specific functions. However, we identified a limited set of genes such as NXF3 and TRIM63, which were up-regulated by the mRNA encoded by the lentiviral transgene. Together, these data confirmed that the CYP2D6 transgenic HepaRG cells represent a suitable optimized transgenic model of HepaRG cells to evaluate biotransformation and toxicity of specific compounds metabolized by CYP2D6.

cell biology↗

bioRxiv2023

Paracetamol/acetaminophen hepatotoxicity: new markers for monitoring the elimination of the reactive N-Acetyl-p-benzoquinone imine

Paracetamol/acetaminophen (N-acetyl-p-aminophenol, APAP) overdose is one of the most important causes of drug-induced liver injury worldwide. Hepatotoxicity induced by APAP is mainly caused by the production of N-acetyl-p-benzoquinone imine (NAPQI), a highly reactive intermediate formed predominantly via the cytochrome P450 2E1. Here, we used human studies and in vitro models to demonstrate that NAPQI-derived thiomethyl metabolites identified using high-resolution mass spectrometry could serve to monitor NAPQI detoxification and elimination in patients (after intake at recommended dose or after intoxication), and to study inter-individual variability in NAPQI production. Using in vitro human models, we showed that these thiomethyl metabolites are directly linked to NAPQI detoxification since they are mainly formed after exposure to glutathione-derived conjugates via an overlooked pathway called the thiomethyl shunt. These long-term thiomethyl metabolites have great potential in future clinical studies in order to provide a more reliable history of APAP ingestion in case of acute intoxication or to study underlying causes involved in APAP-induced hepatotoxicity. One Sentence SummaryThiomethyl metabolites are new markers to monitor the elimination of the toxic N-acetyl-p-benzoquinone imine after therapeutic use or intoxication.

pharmacology and toxicology↗

bioRxiv2022

Adaptation of redox metabolism in drug-tolerant persister cells is a vulnerability to prevent relapse in pancreatic cancer

Pancreatic Ductal Adenocarcinoma (PDAC) remains a major unresolved disease because of its remarkable therapeutic resistance. Even patients who respond to initial therapy experience relapse in most cases. The mechanisms underlying therapy-acquired resistance supporting relapse are poorly understood. In this study, we aimed to determine the metabolic features of PDAC during relapse, specifically adaptations of mitochondrial and redox metabolism. We used preclinical PDAC mouse models (patient-derived xenografts and murine syngeneic allografts) that present complete regression under initial chemotherapeutic treatment but relapse after a certain time. Relapsed tumors were analyzed ex vivo by flow cytometry to measure mitochondrial and redox characteristics. Molecular mechanisms were investigated by quantification of ATP and antioxidants levels, RT-qPCR and bulk RNA-sequencing. Our findings show that mitochondrial metabolism is reprogrammed during relapse, with increased mitochondrial mass, ATP levels, mitochondrial superoxide anions, and total ROS levels, in relapsed compared to control tumors in both models; mitochondrial membrane potential is increased in the xenografts model only. This mitochondrial metabolic reprogramming occurs during treatment-induced regression and at relapse onset. At the molecular level, antioxidant defenses are increased in relapsed tumors and during treatment. These data suggest that treatment-induced oxidative stress may cause the appearance of treatment-adapted cells, known as drug-tolerant persister (DTP) cells. Finally, the combined treatment of arsenic trioxide (ROS inducer) and buthionine sulfoximine (glutathione synthesis inhibitor) is able to completely prevent relapse in PDAC xenografts. In conclusion, targeting redox metabolism via ROS production and antioxidant inhibition is a very promising approach to prevent relapse in PDAC patients. SignificanceMitochondrial and redox metabolisms are reprogrammed during treatment-acquired resistance in pancreatic cancer promoting the survival of drug-tolerant persister cancer cells, opening up new avenues for better therapeutic management of patients.

cancer biology↗

bioRxiv2022

Integrative study of mitochondrial dysfunction in skeletal muscle during pancreatic cancer cachexia

AimsPancreatic Ductal AdenoCarcinoma (PDAC), the most common pancreatic cancer, is a deadly cancer, often diagnosed late and resistant to current therapies. PDAC patients are frequently affected by cachexia characterized by muscle mass and strength loss (sarcopenia) contributing to patient frailty and poor therapeutic response. The objective of this work was to investigate the mechanisms underlying mitochondrial remodeling in the cachectic skeletal muscle, through an integrative study combining functional, morphological and omics-based evaluation of gastrocnemius muscle from genetically-engineered mice developing autochthonous pancreatic tumor and cachexia (KIC GEMM). ResultsKIC cachectic PDAC mice exhibit severe sarcopenia with loss of muscle mass and strength associated with reduced muscle fibers size and induction of protein degradation processes. Mitochondria in PDAC atrophied muscles show reduced respiratory capacities and structural alterations, associated with deregulation of oxidative phosphorylation and mitochondrial dynamics pathways. Beyond the metabolic pathways known to be altered in sarcopenic muscle (carbohydrates, proteins, and redox), lipid and nucleic acid metabolisms are also affected. While the number of mitochondria per cell is not altered, mitochondrial mass is decreased by a factor of 2 and the mitochondrial DNA by a factor of 3, suggesting a defect in mitochondrial genome homeostasis. InnovationMuscle atrophy is associated with strong mitochondrial metabolic defects that are not limited to carbohydrate, protein and redox metabolism, but concern also lipid and nucleic acid metabolism. ConclusionThis work provides a framework to guide towards the most relevant targets in the clinic to limit PDAC-induced cachexia, in particular mitochondrial fatty acid metabolism.

cancer biology↗