Internalization and mechanisms of toxicity of lipid nanocapsules in HepG2 and HepaRG hepatoma cells upon acute and chronic exposures.

Lipid nanocapsules (LNCs) used as nanomedicine have been developed to enhance pharmacokinetics and decrease side effects of drugs, particularly for cancer therapies. After intravenous administration, LNCs possess an important hepatic tropism however, few data exist about their toxicity and even less after repeated exposure. This study aimed to assess the in vitro toxicity and internalization of unloaded LNCs, of 50 and 100 nm size, on HepG2 and HepaRG liver cell lines. Internalization of the 50 nm LNCs was slower compared to the 100 nm LNCs and both LNCs exhibited a higher toxicity on cancerous HepG2 cells compared to differentiated HepaRG cells. LNCs were mainly internalized via caveolin-mediated endocytosis in both cell lines. Upon chronic exposure, the toxicity of LNCs on HepaRG cells increased, although the pathways of internalization remained unchanged. Cell death studies have demonstrated an involvement of ferroptosis, but not of apoptosis. After acute and repeated exposures on HepaRG cells, the 100 nm LNCs showed a good safety profile. Finally, LNCs induced a more significant toxicity associated with faster internalization in the HepG2 cancerous model than in the differentiated HepaRG cells. This provides good evidence for LNCs to potentialize the cytotoxic effects of an active drug on liver cancer cells.

Synthesis, characterization and stability of crosslinked chitosan-maltodextrin pH-sensitive nanogels.

Polysaccharide-based nanogels offer a wide range of chemical compositions and are of great interest due to their biodegradability, biocompatibility, non-toxicity, and their ability to display pH, temperature, or enzymatic response. In this work, we synthesized monodisperse and tunable pH-sensitive nanogels by crosslinking, through reductive amination, chitosan and partially oxidized maltodextrins, by keeping the concentration of chitosan close to its overlap concentration, i.e. in the dilute and semi-dilute regime. The chitosan/maltodextrin nanogels presented sizes ranging from 63 ± 9 to 279 ± 16 nm, showed quasi-spherical and cauliflower-like morphology, reached a ζ-potential of +36 ± 2 mV and maintained a colloidal stability for up to 7 weeks. It was found that the size and surface charge of nanogels depended both on the oxidation degree of maltodextrins and chitosan concentration, as well as on its degree of acetylation and protonation, the latter tuned by pH. The pH-responsiveness of the nanogels was evidenced by an increased size, owed to swelling, and ζ-potential when pH was lowered. Finally, maltodextrin-chitosan biocompatible nanogels were assessed by cell viability assay performed using the HEK293T cell line.

Low concentrations of ethylene bisdithiocarbamate pesticides maneb and mancozeb impair manganese and zinc homeostasis to induce oxidative stress and caspase-dependent apoptosis in human hepatocytes.

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

Identification of Novel Compounds Inhibiting the Kinase Activity of the CDK5/p25 Complex via Direct Binding to p25.

Tamoxifen, the gold standard drug for endocrine therapy for breast cancer, modulates the phosphorylation status of the TAU protein in Alzheimer's disease by inhibiting CDK5 kinase activity. Its binding to p25 prevents CDK5/p25 complexation and hence a decrease of CDK5 activity. In breast tumors, this complex is involved in the proliferation and survival of cancer cells, as well as in the disease's prognosis. Still, the molecular stability of the CDK5/p25 complex following tamoxifen exposure in this cancer type has not yet been clearly deciphered. Here, we report the functional characterization of CDK5 and its p25 regulatory subunit in the absence and presence of tamoxifen. In addition, two novel inhibitors of the kinase activity of the CDK5/p25 complex are identified, both of which would reduce the risk of recurrence of estrogen receptor-positive (ER+) breast cancers and prevent drawbacks induced by tamoxifen exposure. Accordingly, 6His-CDK5 and 6His-p25 have been expressed and purified. Fluorescence anisotropy measurements have been used to assess that the two proteins do form an active complex, and thermodynamic parameters of their interaction were measured. It was also confirmed that tamoxifen directly binds to p25 and inhibits CDK5 kinase activity. Similar observations were obtained using 4-hydroxytamoxifen, an active metabolized form of tamoxifen. Two novel compounds have been identified here that harbor a benzofuran moiety and were shown to target directly p25, and their bindings resulted in decreased CDK5 kinase activity. This encouraging alternative opens the way to the ensuing chemical optimization of this scaffold. It also promises a more specific therapeutic approach that may both tackle the pathological signaling in breast cancer and provide a potential new drug for Alzheimer's disease.

Liposome-Mediated Gene Transfer in Differentiated HepaRG™ Cells: Expression of Liver Specific Functions and Application to the Cytochrome P450 2D6 Expression.

The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 and progenitor HepaRG™ human hepatoma cells. The lipophosphoramidate Syn1-based nanovector, which allowed the highest transfection efficiencies of progenitor HepaRG™ cells, was next used to transfect differentiated HepaRG™ cells. Lipofection of these cells using Syn1-based liposome was poorly efficient most likely because the differentiated HepaRG™ cells are highly quiescent. Thus, we engineered the differentiated HepaRG™ Mitogenic medium supplement (ADD1001) that triggered robust proliferation of differentiated cells. Importantly, we characterized the phenotypical changes occurring during proliferation of differentiated HepaRG™ cells and demonstrated that mitogenic stimulation induced a partial and transient decrease in the expression levels of some liver specific functions followed by a fast recovery of the full differentiation status upon removal of the mitogens. Taking advantage of the proliferation of HepaRG™ cells, we defined lipofection conditions using Syn1-based liposomes allowing transient expression of the cytochrome P450 2D6, a phase I enzyme poorly expressed in HepaRG cells, which opens new means for drug metabolism studies in HepaRG™ cells.

Hepatotropic Peptides Grafted onto Maleimide-Decorated Nanoparticles: Preparation, Characterization and In Vitro Uptake by Human HepaRG Hepatoma Cells.

We recently demonstrated the strong tropism of George Baker (GB) Virus A (GBVA10-9) and Plasmodium circumsporozoite protein (CPB) derived synthetic peptides towards hepatoma cells. In a first approach, these peptides were covalently bound to poly(benzyl malate) (PMLABe73) and poly(ethylene glycol)-block-PMLABe73 (PEG62-b-PMLABe73) (co)polymers, and corresponding peptide-decorated nanoparticles (NPs) were prepared by nanoprecipitation. We showed that peptide enhanced NPs internalization by hepatoma cells. In the present work, we set up a second strategy to functionalize NPs prepared from PMLABe73 derivates. First, maleimide-functionalized PMLABe73 (Mal-PMLABe73) and PEG62-b-PMLABe73 (Mal-PEG62-b-PMLABe73) were synthesized and corresponding NPs were prepared by nanoprecipitation. Then, peptides (GBVA10-9, CPB and their scramble controls GBVA10-9scr and CPBscr) with a thiol group were engrafted onto the NPs' maleimide groups using the Michael addition to obtain peptide functionalized NPs by post-formulation procedure. These peptide-modified NPs varied in diameter and dispersity depending on the considered peptides and/or (co)polymers but kept their spherical shape. The peptide-functionalized NPs were more efficiently internalized by HepaRG hepatoma cells than native and maleimide-NPs with various levels relying on the peptide's nature and the presence of PEG. We also observed important differences in internalization of NPs functionalized by the maleimide-thiol-peptide reaction compared to that of NPs prepared from peptide-functionalized PMLABe73 derivatives.

Circumsporozoite Protein of Plasmodium berghei- and George Baker Virus A-Derived Peptides Trigger Efficient Cell Internalization of Bioconjugates and Functionalized Poly(ethylene glycol)-b-poly(benzyl malate)-Based Nanoparticles in Human Hepatoma Cells.

In order to identify the peptides, selected from the literature, that exhibit the strongest tropism towards human hepatoma cells, cell uptake assays were performed using biotinylated synthetic peptides bound to fluorescent streptavidin or engrafted onto nanoparticles (NPs), prepared from biotin-poly(ethylene glycol)-block-poly(benzyl malate) (Biot-PEG-b-PMLABe) via streptavidin bridging. Two peptides, derived from the circumsporozoite protein of Plasmodium berghei- (CPB) and George Baker (GB) Virus A (GBVA10-9), strongly enhanced the endocytosis of both streptavidin conjugates and NPs in hepatoma cells, compared to primary human hepatocytes and non-hepatic cells. Unexpectedly, the uptake of CPB- and GBVA10-9 functionalized PEG-b-PMLABe-based NPs by hepatoma cells involved, at least in part, the peptide binding to apolipoproteins, which would promote NP's interactions with cell membrane receptors of HDL particles. In addition, CPB and GBVA10-9 peptide-streptavidin conjugates favored the uptake by hepatoma cells over that of the human macrophages, known to strongly internalize nanoparticles by phagocytosis. These two peptides are promising candidate ligands for targeting hepatocellular carcinomas.

Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition.

(1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

Synthesis of Poly(Malic Acid) Derivatives End-Functionalized with Peptides and Preparation of Biocompatible Nanoparticles to Target Hepatoma Cells.

Recently, short synthetic peptides have gained interest as targeting agents in the design of site-specific nanomedicines. In this context, our work aimed at developing new tools for the diagnosis and/or therapy of hepatocellular carcinoma (HCC) by grafting the hepatotropic George Baker (GB) virus A (GBVA10-9) and Plasmodium circumsporozoite protein (CPB)-derived peptides to the biocompatible poly(benzyl malate), PMLABe. We successfully synthesized PMLABe derivatives end-functionalized with peptides GBVA10-9, CPB, and their corresponding scrambled peptides through a thiol/maleimide reaction. The corresponding nanoparticles (NPs), varying by the nature of the peptide (GBVA10-9, CPB, and their scrambled peptides) and the absence or presence of poly(ethylene glycol) were also successfully formulated using nanoprecipitation technique. NPs were further characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS) and transmission electron microscopy (TEM), highlighting a diameter lower than 150 nm, a negative surface charge, and a more or less spherical shape. Moreover, a fluorescent probe (DiD Oil) has been encapsulated during the nanoprecipitation process. Finally, preliminary in vitro internalisation assays using HepaRG hepatoma cells demonstrated that CPB peptide-functionalized PMLABe NPs were efficiently internalized by endocytosis, and that such nanoobjects may be promising drug delivery systems for the theranostics of HCC.

Overview of Radiolabeled Somatostatin Analogs for Cancer Imaging and Therapy.

Identified in 1973, somatostatin (SST) is a cyclic hormone peptide with a short biological half-life. Somatostatin receptors (SSTRs) are widely expressed in the whole body, with five subtypes described. The interaction between SST and its receptors leads to the internalization of the ligand-receptor complex and triggers different cellular signaling pathways. Interestingly, the expression of SSTRs is significantly enhanced in many solid tumors, especially gastro-entero-pancreatic neuroendocrine tumors (GEP-NET). Thus, somatostatin analogs (SSAs) have been developed to improve the stability of the endogenous ligand and so extend its half-life. Radiolabeled analogs have been developed with several radioelements such as indium-111, technetium-99 m, and recently gallium-68, fluorine-18, and copper-64, to visualize the distribution of receptor overexpression in tumors. Internal metabolic radiotherapy is also used as a therapeutic strategy (e.g., using yttrium-90, lutetium-177, and actinium-225). With some radiopharmaceuticals now used in clinical practice, somatostatin analogs developed for imaging and therapy are an example of the concept of personalized medicine with a theranostic approach. Here, we review the development of these analogs, from the well-established and authorized ones to the most recently developed radiotracers, which have better pharmacokinetic properties and demonstrate increased efficacy and safety, as well as the search for new clinical indications.

Alteration of immunophenotype of human macrophages and monocytes after exposure to cigarette smoke.

Cigarette smoke exposure (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD). Macrophages have an important role in COPD because they release pro-inflammatory and anti-inflammatory cytokines. The present study's we investigate the functional changes in macrophages and monocytes exposed to cigarette smoke extract (CSE). Herein, using human monocyte-derived macrophages (MDMs) from healthy donors and we found that CSE was not associated with significant changes in the production of pro inflammatory cytokines by MDMs. In contrast, exposure to CSE suppressed the production of IL-6 and Gro-a/CXCL1 by LPS-stimulated-MDMs, but had an additive effect on the release of IL-8/CXCL8 and MCP1/CCL2. However, CSE exposure was associated with greater production, TARC/CCL-17 and CCL22/MDC. Moreover, MDMs displayed a lower uptake capacity after CSE exposure. We identify, for what is to our knowledge the first time that monocytes from patients with COPD produced less IL-8/CXCL8 and Gro-α/CXCL1 after LPS stimulation and produced higher levels of TARC/CCL17 and MDC/CCL-22 after IL-4 stimulation. Our present results highlighted a skewed immune response, with an imbalance in M1 vs. M2 cytokine production. In conclusion, exposure to CS has contrasting, multifaceted effects on macrophages and monocytes. Our data may provide a better understanding of the mechanisms underlying COPD.

Synthesis of Poly(Dimethylmalic Acid) Homo- and Copolymers to Produce Biodegradable Nanoparticles for Drug Delivery: Cell Uptake and Biocompatibility Evaluation in Human Heparg Hepatoma Cells.

Hydrophobic and amphiphilic derivatives of the biocompatible and biodegradable poly(dimethylmalic acid) (PdiMeMLA), varying by the nature of the lateral chains and the length of each block, respectively, have been synthesized by anionic ring-opening polymerization (aROP) of the corresponding monomers using an initiator/base system, which allowed for very good control over the (co)polymers' characteristics (molar masses, dispersity, nature of end-chains). Hydrophobic and core-shell nanoparticles (NPs) were then prepared by nanoprecipitation of hydrophobic homopolymers and amphiphilic block copolymers, respectively. Negatively charged NPs, showing hydrodynamic diameters (Dh) between 50 and 130 nm and narrow size distributions (0.08 < PDI < 0.22) depending on the (co)polymers nature, were obtained and characterized by dynamic light scattering (DLS), zetametry, and transmission electron microscopy (TEM). Finally, the cytotoxicity and cellular uptake of the obtained NPs were evaluated in vitro using the hepatoma HepaRG cell line. Our results showed that both cytotoxicity and cellular uptake were influenced by the nature of the (co)polymer constituting the NPs.

Roles of CDK/Cyclin complexes in transcription and pre-mRNA splicing: Cyclins L and CDK11 at the cross-roads of cell cycle and regulation of gene expression.

Cyclin Dependent Kinases (CDKs) represent a large family of serine/threonine protein kinases that become active upon binding to a Cyclin regulatory partner. CDK/cyclin complexes recently identified, as well as "canonical" CDK/Cyclin complexes regulating cell cycle, are implicated in the regulation of gene expression via the phosphorylation of key components of the transcription and pre-mRNA processing machineries. In this review, we summarize the role of CDK/cyclin-dependent phosphorylation in the regulation of transcription and RNA splicing and highlight recent findings that indicate the involvement of CDK11/cyclin L complexes at the cross-roads of cell cycle, transcription and RNA splicing. Finally, we discuss the potential of CDK11 and Cyclins L as therapeutic targets in cancer.

Non-oxidative ethanol metabolism in human hepatic cells in vitro: Involvement of uridine diphospho-glucuronosyltransferase 1A9 in ethylglucuronide production.

Ethanol is the most frequently psychoactive substance used in the world, leading to major public health problems with several millions of deaths attributed to alcohol consumption each year. Metabolism of ethanol occurs mainly in the liver via the predominant oxidative metabolism pathway involving phase I enzymes including alcohol dehydrogenases (ADH), cytochrome P450 (CYP) 2E1 and catalase. In a lesser extent, an alternative non-oxidative pathway also contributes to the metabolism of ethanol, which involves the uridine diphospho-glucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II enzymes. Using liquid chromatography-high resolution mass spectrometry, ethylglucuronide (EtG) and ethylsulfate (EtS) produced respectively by UGT and SULT conjugation and detected in various biological samples are direct markers of alcohol consumption. We report herein the efficient non-oxidative metabolic pathway of ethanol in human differentiated HepaRG cells compared to primary human hepatocytes (HH). We showed dose- and time-dependent production of EtS and EtG after ethanol (25 or 50 mM) treatment in culture media of differentiated HepaRG cells and HH and a significant induction of CYP2E1 mRNA expression upon acute ethanol exposure in HepaRG cells. These differentiated hepatoma cells thus represent a suitable in vitro human liver cell model to explore ethanol metabolism and more particularly EtG and EtS production. In addition, using recombinant HepG2 cells expressing different UGT1A genes, we found that UGT1A9 was the major UGT involved in ethanol glucuronidation.

Cytochrome P450 1A1/2, 2B6 and 3A4 HepaRG Cell-Based Biosensors to Monitor Hepatocyte Differentiation, Drug Metabolism and Toxicity.

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.

Bulk Organocatalytic Synthetic Access to Statistical Copolyesters from l-Lactide and ε-Caprolactone Using Benzoic Acid.

The development of synthetic strategies to produce statistical copolymers based on l-lactide (l-LA) and ε-caprolactone (CL), denoted as P(LA- stat-CL), remains highly challenging in polymer chemistry. This is due to the differing reactivity of the two monomers during their ring-opening copolymerization (ROcP). Yet, P(LA- stat-CL) materials are highly sought after as they combine the properties of both polylactide (PLA) and poly(ε-caprolactone) (PCL). Here, benzoic acid (BA), a naturally occurring, cheap, readily recyclable, and thermally stable weak acid, is shown to trigger the organocatalyzed ring-opening copolymerization (OROcP) of l-LA and CL under solvent-free conditions at 155 °C, in presence of various alcohols as initiators, with good control over molar masses and dispersities (1.11 < D < 1.35) of the resulting copolyesters. Various compositions can be achieved, and the formation of statistical compounds is shown through characterization by 1H, 13C, and diffusion ordered spectroscopy NMR spectroscopies and by differential scanning calorimetry, as well as through the determination of reactivity ratios ( rLA = 0.86, rCL = 0.86), using the visualization of the sum of squared residuals space method. Furthermore, this BA-OROcP process can be exploited to access metal-free PLA- b-P(LA- stat-CL)- b-PLA triblock copolymers, using a diol as an initiator. Finally, residual traces of BA remaining in P(LA- stat-CL) copolymers (<0.125 mol %) do not show any cytotoxicity toward hepatocyte-like HepaRG cells, demonstrating the safety of this organic catalyst.

Ethanol upregulates the P2X7 purinergic receptor in human macrophages.

Alcohol consumption is considered to be the third leading cause of death in the United States. In addition to its direct toxicity, ethanol has two contrasting effects on the immune system: the nucleotide oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome is inhibited by acute ethanol exposure but activated by chronic ethanol exposure. Purinergic receptors (especially the P2X7 receptor) are able to activate the NLRP3 inflammasome and are involved in many ethanol-related diseases (such as gout, pulmonary fibrosis, alcoholic steatohepatitis, and certain cancers). We hypothesized that ethanol regulates purinergic receptors and thus modulates the NLRP3 inflammasome's activity. In experiments with monocyte-derived macrophages, we found that interleukin (IL)-1ß secretion was inhibited after 7 h of exposure (but not 48 h of exposure) to ethanol. The disappearance of ethanol's inhibitory effect on IL-1ß secretion after 48 h was not mediated by the upregulated production of IL-1ß, IL-1α, IL-6 or the inflammasome components NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase 1. P2X7R expression was upregulated by ethanol, whereas expression of the P2X4 and P2X1 receptors was not. Taken as a whole, our results suggest that ethanol induces NLRP3 inflammasome activation by upregulating the P2X7 receptor. This observation might have revealed a new mechanism for inflammation in ethanol-related diseases.

Production of chlorzoxazone glucuronides via cytochrome P4502E1 dependent and independent pathways in human hepatocytes.

CYP2E1 activity is measured in vitro and in vivo via hydroxylation of the Chlorzoxazone (CHZ) producing the 6-hydroxychlorzoxazone (OH-CHZ) further metabolized as a glucuronide excreted in urine. Thus, the quantification of the OH-CHZ following enzymatic hydrolysis of CHZ-derived glucuronide appears to be a reliable assay to measure the CYP2E1 activity without direct detection of this glucuronide. However, OH-CHZ hydrolyzed from urinary glucuronide accounts for less than 80% of the CHZ administrated dose in humans leading to postulate the production of other unidentified metabolites. Moreover, the Uridine 5'-diphospho-glucuronosyltransferase (UGT) involved in the hepatic glucuronidation of OH-CHZ has not yet been identified. In this study, we used recombinant HepG2 cells expressing CYP2E1, metabolically competent HepaRG cells, primary hepatocytes and precision-cut human liver slices to identify metabolites of CHZ (300 µM) by high pressure liquid chromatography-UV and liquid-chromatography-mass spectrometry analyses. Herein, we report the detection of the CHZ-O-glucuronide (CHZ-O-Glc) derived from OH-CHZ in culture media but also in mouse and human urine and we identified a novel CHZ metabolite, the CHZ-N-glucuronide (CHZ-N-Glc), which is resistant to enzymatic hydrolysis and produced independently of CHZ hydroxylation by CYP2E1. Moreover, we demonstrate that UGT1A1, 1A6 and 1A9 proteins catalyze the synthesis of CHZ-O-Glc while CHZ-N-Glc is produced by UGT1A9 specifically. Together, we demonstrated that hydrolysis of CHZ-O-Glc is required to reliably quantify CYP2E1 activity because of the rapid transformation of OH-CHZ into CHZ-O-Glc and identified the CHZ-N-Glc produced independently of the CYP2E1 activity. Our results also raise the questions of the contribution of CHZ-N-Glc in the overall CHZ metabolism and of the quantification of CHZ glucuronides in vitro and in vivo for measuring UGT1A activities.

Dosimetric parameters predicting contralateral liver hypertrophy after unilobar radioembolization of hepatocellular carcinoma.

PURPOSE: This study aimed at identifying prior therapy dosimetric parameters using 99mTc-labeled macro-aggregates of albumin (MAA) that are associated with contralateral hepatic hypertrophy occurring after unilobar radioembolization of hepatocellular carcinoma (HCC) performed with 90Y-loaded glass microspheres. METHODS: The dosimetry data of 73 HCC patients were collected prior to the treatment with 90Y-loaded microspheres for unilateral disease. The injected liver dose (ILD), the tumor dose (TD) and healthy injected liver dose (HILD) were calculated based on MAA quantification. Following treatment, the maximal hypertrophy (MHT) of an untreated lobe was calculated. RESULTS: Mean MHT was 35.4 ± 40.4%. When using continuous variables, the MHT was not correlated with any tested variable, i.e., injected activity, ILD, HILD or TD except with a percentage of future remnant liver (FRL) following the 90Y-microspheres injection  (r = -0.56). MHT ≥ 10% was significantly more frequent for patients with HILD ≥ 88 Gy, (52% of the cases), i.e., in 92.2% versus 65.7% for HILD < 88 Gy (p = 0.032). MHT ≥ 10% was also significantly more frequent for patients with a TD ≥ 205 Gy and a tumor volume (VT) ≥ 100 cm3 in patients with initial FRL < 50%. MHT ≥10% was seen in 83.9% for patients with either an HILD ≥ 88 Gy or a TD ≥ 205 Gy for tumors larger than 100cm3 (85% of the cases), versus only 54.5% (p = 0.0265) for patients with none of those parameters. MHT ≥10% was also associated with FRL and the Child-Pugh score. Using multivariate analysis, the Child-Pugh score (p < 0.0001), FRL (p = 0.0023) and HILD (p = 0.0029) were still significantly associated with MHT ≥10%. CONCLUSION: This study demonstrates for the first time that HILD is significantly associated with liver hypertrophy. There is also an impact of high tumor doses in large lesions in one subgroup of patients. Larger prospective studies evaluating the MAA dosimetric parameters have to be conducted to confirm these promising results.

Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages.

The design of drug-loaded nanoparticles (NPs) appears to be a suitable strategy for the prolonged plasma concentration of therapeutic payloads, higher bioavailability, and the reduction of side effects compared with classical chemotherapies. In most cases, NPs are prepared from (co)polymers obtained through chemical polymerization. However, procedures have been developed to synthesize some polymers via enzymatic polymerization in the absence of chemical initiators. The aim of this work was to compare the acute in vitro cytotoxicities and cell uptake of NPs prepared from poly(benzyl malate) (PMLABe) synthesized by chemical and enzymatic polymerization. Herein, we report the synthesis and characterization of eight PMLABe-based polymers. Corresponding NPs were produced, their cytotoxicity was studied in hepatoma HepaRG cells, and their uptake by primary macrophages and HepaRG cells was measured. In vitro cell viability evidenced a mild toxicity of the NPs only at high concentrations/densities of NPs in culture media. These data did not evidence a higher biocompatibility of the NPs prepared from enzymatic polymerization, and further demonstrated that chemical polymerization and the nanoprecipitation procedure led to biocompatible PMLABe-based NPs. In contrast, NPs produced from enzymatically synthesized polymers were more efficiently internalized than NPs produced from chemically synthesized polymers. The efficient uptake, combined with low cytotoxicity, indicate that PMLABe-based NPs are suitable nanovectors for drug delivery, deserving further evaluation in vivo to target either hepatocytes or resident liver macrophages.