Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay

International audienceMany efforts have been made in the last 30 years to develop more relevant in vitro models to study genotoxic responses of drugs and environmental contaminants. While 2D HepaRG cells are one of the most promising models for liver toxicology, a switch to 3D cultures that integrate both in vivo architecture and cell-cell interactions has occurred to achieve even more predictive models. preliminary studies have indicated that 3D HepaRG cells are suitable for liver toxicity screening. Our study aimed to evaluate the response of HepaRG spheroids exposed to various genotoxic compounds using the single cell gel electrophoresis assay. HepaRG spheroids were used at 10 days after seeding and exposed for 24 and 48 hours to certain selected chemical compounds (methylmethansulfonate (MMS), etoposide, benzo[a]pyrene (B[a]P), cyclophosphamide (CPA), 7,12-dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), 4-nitroquinoline (4-NQO), 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), acrylamide, and 2-4-diaminotoluene (2,4-DAT)). After treatment, the comet assay was performed on single cell suspensions and cytotoxicity was determined by the ATP assay. Comet formation was observed for all compounds except IQ, etoposide and 2,4-DAT. Treatment of spheroids with rifampicin increased CYP3A4 activity, demonstrating the metabolic capacity of HepaRG spheroids. These data on genotoxicity in 3D HepaRG spheroids are promising, but further experiments are required to prove that this model can improve the predictivity of in vitro models to detect human carcinogens

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.The European Partnership for the Assessment of Risks from Chemicals has received funding from the European Union’s Horizon Europe research and innovation program under Grant Agreement No 101057014 and has received co-funding of the authors’ institutions. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.info:eu-repo/semantics/publishedVersio

Impact of solvents on the in vitro genotoxicity of TMPTA in human HepG2 cells

International audienceSmall hydrophobic chemical compounds require solvents to produce suitable solutions for toxicological studies. However, some solvents can modify the biological properties of substances and therefore their toxicity. This specific issue has been raised for PEG-400 as an anti-inflammatory and anti-oxidative compound. Recently, in the context of the REACH Regulation, PEG-400 was used to test the in vivo genotoxicity of trimethylolpropane triacrylate (TMPTA) in the comet assay. TMPTA failed to increase DNA damage whereas it induces genotoxicity in vitro in DMSO. Therefore, we questioned whether PEG-400 could modify the genotoxicity of TMPTA. The aim of this study was to determine the potential impact of PEG-400 on the in vitro genotoxicity of TMPTA, compared to DMSO. TMPTA was dissolved in either PEG-400 or DMSO, and the induction of γH2AX and Caspase-3 was analyzed in HepG2 cells. TMPTA induced γH2AX and Caspase-3 with both PEG-400 and DMSO. However, TMPTA induced effects at 4-fold lower concentrations when PEG-400 is used as the solvent compared to DMSO. While genotoxic effects are observed at much lower concentrations with PEG-400, it does not modify the in vitro genotoxicity of TMPTA. However, further in vitro studies with small hydrophobic compounds should be done to clarify the effect of PEG-400. Moreover, in vivo studies should be performed to confirm that PEG-400 remains suitable for in vivo genotoxicity tests

Metabolism and bioactivation of 2-amino-9H-pyrido[2,3-b]indole, a tobacco carcinogen, in human hepatocytes

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Mixtures of Lipophilic Phycotoxins: Exposure Data and Toxicological Assessment

International audienceLipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species. They accumulate in filter-feeding shellfish and can cause human intoxication. Regulatory limits have been set for individual toxins, and the toxicological features are well characterized for some of them. However, phycotoxin contamination is often a co-exposure phenomenon, and toxicological data regarding mixtures effects are very scarce. Moreover, the type and occurrence of phycotoxins can greatly vary from one region to another. This review aims at summarizing the knowledge on (i) multi-toxin occurrence by a comprehensive literature review and (ii) the toxicological assessment of mixture effects. A total of 79 publications was selected for co-exposure evaluation, and 44 of them were suitable for toxin ratio calculations. The main toxin mixtures featured okadaic acid in combination with pectenotoxin-2 or yessotoxin. Only a few toxicity studies dealing with co-exposure were published. In vivo studies did not report particular mixture effects, whereas in vitro studies showed synergistic or antagonistic effects. Based on the combinations that are the most reported, further investigations on mixture effects must be carried out

Metabolism of the Tobacco Carcinogen 2-Amino-9H-pyrido[2,3-b]indole (AalphaC) in Primary Human Hepatocytes

International audience2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant carcinogenic heterocyclic aromatic amine (HAA) formed in mainstream tobacco smoke. AαC is a liver carcinogen in rodents, but its carcinogenic potential in humans is not known. To obtain a better understanding of the genotoxicity of AαC in humans, we have investigated its metabolism and its ability to form DNA adducts in human hepatocytes. Primary human hepatocytes were treated with AαC at doses ranging from 0.1-50 μM, and the metabolites were characterized by ultra-performance LC/ion trap multistage mass spectrometry (UPLC/MS). Six major metabolites were identified: a ring-oxidized doubly conjugated metabolite, N-acetyl-2-amino-9H-pyrido[2,3-b]indole-6-yl-oxo-(β-d-glucuronic acid) (N-acetyl-AαC-6-O-Gluc); two ring-oxidized glucuronide (Gluc) conjugates: 2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(β-d-glucuronic acid) (AαC-3-O-Gluc) and 2-amino-9H-pyrido[2,3-b]indol-6-yl-oxo-(β-d-glucuronic acid) (AαC-6-O-Gluc); two sulfate conjugates, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (AαC-3-O-SOH) and 2-amino-9H-pyrido[2,3-b]indol-6-yl sulfate (AαC-6-O-SOH); and the Gluc conjugate, N-(β-d-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N-Gluc). In addition, four minor metabolites were identified: N-acetyl-9H-pyrido[2,3-b]indol-3-yl sulfate (N-acetyl-AαC-3-O-SOH), N-acetyl-9H-pyrido[2,3-b]indol-6-yl sulfate (N-acetyl-AαC-6-O-SOH), N-acetyl-2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(β-d-glucuronic acid) (N-acetyl-AαC-3-O-Gluc), and O-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN-O-Gluc). The latter metabolite, AαC-HN-O-Gluc is a reactive intermediate that binds to DNA to form the covalent adduct N-(2'-deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-AαC). Preincubation of hepatocytes with furafylline, a selective mechanism-based inhibitor of P450 1A2, resulted in a strong decrease in the formation of AαC-HN-O-Gluc and a concomitant decrease in DNA adduct formation. Our findings describe the major pathways of metabolism of AαC in primary human hepatocytes and reveal the importance of N-acetylation and glucuronidation in metabolism of AαC. P450 1A2 is a major isoform involved in the bioactivation of AαC to form the reactive AαC-HN-O-Gluc conjugate and AαC-DNA adducts

Identification of key pathways involved in the toxic response of the cyanobacterial toxin cylindrospermopsin in human hepatic HepaRG cells

International audienceThe hepatotoxin cylindrospermopsin (CYN) has been involved in cases of poisoning in humans following ingestion. As its liver toxicity process is complex, we studied the transcriptomic profile of HepaRG cells exposed to CYN. The affected pathways were confirmed through the expression of key genes and the investigation of toxicity markers. In addition, CYP450 activities and cell redox homeostasis were investigated following acute and repeated exposure. CYN induced the down-regulation of genes involved in xenobiotic metabolism and cell cycle progression. There was cell cycle disturbance characterised by an accumulation of G1/S and G2/M cells and an increase in phospho-H3-positive cells. This was linked to the induction of DNA damage demonstrated by an increase in γH2AX-positive cells as well as an accumulation of sub-G1 cells indicating apoptosis but not involving caspase-3. While glutathione (GSH) content sharply decreased following acute exposure to CYN, it increased following repeated exposure, reflecting an adaptive response of cell redox homeostasis. However, our data also suggested that CYN induced the down-regulation of phase I and II metabolism gene products, and CYP450 activities were affected following both acute and repeated exposure to CYN. Our study indicated that repeated exposure of liver cells to low concentrations of CYN may affect their detoxification capacities

Comparative in silico prediction of P-glycoprotein-mediated transport for 2010-2020 US FDA-approved drugs using six Web-tools

International audienceP-glycoprotein (P-gp) is an efflux pump implicated in pharmacokinetics and drug-drug interactions. The identification of its substrates is consequently an important issue, notably for drugs under development. For such a purpose, various in silico methods have been developed, but their relevance remains to be fully established. The present study was designed to get insight about this point, through determining the performance values of six freely accessible Web-tools (ADMETlab, AdmetSAR2.0, PgpRules, pkCSM, SwissADME and vNN-ADMET), computationally predicting P-gp-mediated transport. Using an external test set of 231 marketed drugs, approved over the 2010-2020 period by the US Food and Drug Administration and fully in vitro characterized for their P-gp substrate status, various performance parameters (including sensitivity, specificity, accuracy, Matthews correlation coefficient and area under the receiver operating characteristics curve) were determined. They were found to rather poorly meet criteria commonly required for acceptable prediction, whatever the Web-tools were used alone or in combination. Predictions of being P-gp substrate or non-substrate by these online in silico methods may therefore be considered with caution

An Evaluation of the Cytotoxic and Genotoxic Effects of the Marine Toxin C17-SAMT in Human TK6 and HepaRG Cell Lines

This study investigates the genotoxicity and cytotoxicity of C17-sphinganine analog mycotoxin (C17-SAMT) using in vitro assays. C17-SAMT was previously identified as the cause of unusual toxicity in cultured mussels from the Bizerte Lagoon in northern Tunisia. While a previous in vivo genotoxicity study was inconclusive, in vitro results demonstrated that C17-SAMT induced an increase in micronucleus formation in human lymphoblastoid TK6 cells at concentrations of 0.87 µM and 1.74 µM. In addition, multiparametric cytotoxicity assays were performed in the human hepatoma HepaRG cell line, which showed that C17-SAMT induced mitochondrial dysfunction, decreased cellular ATP levels, and altered the expression of various proteins, including superoxide dismutase SOD2, heme oxygenase HO-1, and NF-κB. These results suggest that C17-SAMT is mutagenic in vitro and can induce mitochondrial dysfunction in HepaRG cells. However, the exact mode of action of this toxin requires further investigation. Overall, this study highlights the potential toxicity of C17-SAMT and the need for further research to better understand its effects