DMSO-free highly differentiated HepaRG spheroids for chronic toxicity, liver functions and genotoxicity studies.

The liver is essential in the elimination of environmental and food contaminants. Given the interspecies differences between rodents and humans, the development of relevant in vitro human models is crucial to investigate liver functions and toxicity in cells that better reflect pathophysiological processes. Classically, the differentiation of the hepatic HepaRG cell line requires high concentration of dimethyl sulfoxide (DMSO), which restricts its usefulness for drug-metabolism studies. Herein, we describe undifferentiated HepaRG cells embedded in a collagen matrix in DMSO-free conditions that rapidly organize into polarized hollow spheroids of differentiated hepatocyte-like cells (Hepoid-HepaRG). Our conditions allow concomitant proliferation with high levels of liver-specific functions and xenobiotic metabolism enzymes expression and activities after a few days of culture and for at least 4 weeks. By studying the toxicity of well-known injury-inducing drugs by treating cells with 1- to 100-fold of their plasmatic concentrations, we showed appropriate responses and demonstrate the sensitivity to drugs known to induce various degrees of liver injury. Our results also demonstrated that the model is well suited to estimate cholestasis and steatosis effects of drugs following chronic treatment. Additionally, DNA alterations caused by four genotoxic compounds (Aflatoxin B1 (AFB1), Benzo[a]Pyrene (B[a]P), Cyclophosphamide (CPA) and Methyl methanesulfonate (MMS)) were quantified in a dose-dependent manner by the comet and micronucleus assays. Their genotoxic effects were significantly increased after either an acute 24 h treatment (AFB1: 1.5-6 µM, CPA: 2.5-10 µM, B[a]P: 12.5-50 µM, MMS: 90-450 µM) or after a 14-day treatment at much lower concentrations (AFB1: 0.05-0.2 µM, CPA: 0.125-0.5 µM, B[a]P: 0.125-0.5 µM) representative to human exposure. Altogether, the DMSO-free 3D culture of Hepoid-HepaRG provides highly differentiated and proliferating cells relevant for various toxicological in vitro assays, especially for drug-preclinical studies and environmental chemicals risk assessment.

Constructing xenobiotic maps of metabolism to predict enzymes catalyzing metabolites capable of binding to DNA.

BACKGROUND: The liver plays a major role in the metabolic activation of xenobiotics (drugs, chemicals such as pollutants, pesticides, food additives...). Among environmental contaminants of concern, heterocyclic aromatic amines (HAA) are xenobiotics classified by IARC as possible or probable carcinogens (2A or 2B). There exist little information about the effect of these HAA in humans. While HAA is a family of more than thirty identified chemicals, the metabolic activation and possible DNA adduct formation have been fully characterized in human liver for only a few of them (MeIQx, PhIP, A[Formula: see text]C). RESULTS: We have developed a modeling approach in order to predict all the possible metabolites of a xenobiotic and enzymatic profiles that are linked to the production of metabolites able to bind DNA. Our prediction of metabolites approach relies on the construction of an enriched and annotated map of metabolites from an input metabolite.The pipeline assembles reaction prediction tools (SyGMa), sites of metabolism prediction tools (Way2Drug, SOMP and Fame 3), a tool to estimate the ability of a xenobotics to form DNA adducts (XenoSite Reactivity V1), and a filtering procedure based on Bayesian framework. This prediction pipeline was evaluated using caffeine and then applied to HAA. The method was applied to determine enzymes profiles associated with the maximization of metabolites derived from each HAA which are able to bind to DNA. The classification of HAA according to enzymatic profiles was consistent with their chemical structures. CONCLUSIONS: Overall, a predictive toxicological model based on an in silico systems biology approach opens perspectives to estimate the genotoxicity of various chemical classes of environmental contaminants. Moreover, our approach based on enzymes profile determination opens the possibility of predicting various xenobiotics metabolites susceptible to bind to DNA in both normal and physiopathological situations.

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

2-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/MSn). Six major metabolites were identified: a ring-oxidized doubly conjugated metabolite, N2-acetyl-2-amino-9H-pyrido[2,3-b]indole-6-yl-oxo-(ß-d-glucuronic acid) (N2-acetyl-AαC-6-O-Gluc); two ring-oxidized glucuronide (Gluc) conjugates: 2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(ß-d-glucuronic acid) (AαC-3-O-Gluc) and 2-amino-9H-pyrido[2,3-b]indol-6-yl-oxo-(ß-d-glucuronic acid) (AαC-6-O-Gluc); two sulfate conjugates, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (AαC-3-O-SO3H) and 2-amino-9H-pyrido[2,3-b]indol-6-yl sulfate (AαC-6-O-SO3H); and the Gluc conjugate, N2-(ß-d-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N2-Gluc). In addition, four minor metabolites were identified: N2-acetyl-9H-pyrido[2,3-b]indol-3-yl sulfate (N2-acetyl-AαC-3-O-SO3H), N2-acetyl-9H-pyrido[2,3-b]indol-6-yl sulfate (N2-acetyl-AαC-6-O-SO3H), N2-acetyl-2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(ß-d-glucuronic acid) (N2-acetyl-AαC-3-O-Gluc), and O-(ß-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN2-O-Gluc). The latter metabolite, AαC-HN2-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-HN2-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-HN2-O-Gluc conjugate and AαC-DNA adducts.

2-Amino-9H-pyrido[2,3-b]indole (AαC) Adducts and Thiol Oxidation of Serum Albumin as Potential Biomarkers of Tobacco Smoke.

2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine formed during the combustion of tobacco. AαC undergoes bioactivation to form electrophilic N-oxidized metabolites that react with DNA to form adducts, which can lead to mutations. Many genotoxicants and toxic electrophiles react with human serum albumin (albumin); however, the chemistry of reactivity of AαC with proteins has not been studied. The genotoxic metabolites, 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC), 2-nitroso-9H-pyrido[2,3-b]indole (NO-AαC), N-acetyloxy-2-amino-9H-pyrido[2,3-b]indole (N-acetoxy-AαC), and their [(13)C6]AαC-labeled homologues were reacted with albumin. Sites of adduction of AαC to albumin were identified by data-dependent scanning and targeted bottom-up proteomics approaches employing ion trap and Orbitrap MS. AαC-albumin adducts were formed at Cys(34), Tyr(140), and Tyr(150) residues when albumin was reacted with HONH-AαC or NO-AαC. Sulfenamide, sulfinamide, and sulfonamide adduct formation occurred at Cys(34) (AαC-Cys(34)). N-Acetoxy-AαC also formed an adduct at Tyr(332). Albumin-AαC adducts were characterized in human plasma treated with N-oxidized metabolites of AαC and human hepatocytes exposed to AαC. High levels of N-(deoxyguanosin-8-yl)-AαC (dG-C8-AαC) DNA adducts were formed in hepatocytes. The Cys(34) was the sole amino acid of albumin to form adducts with AαC. Albumin also served as an antioxidant and scavenged reactive oxygen species generated by metabolites of AαC in hepatocytes; there was a strong decrease in reduced Cys(34), whereas the levels of Cys(34) sulfinic acid (Cys-SO2H), Cys(34)-sulfonic acid (Cys-SO3H), and Met(329) sulfoxide were greatly increased. Cys(34) adduction products and Cys-SO2H, Cys-SO3H, and Met(329) sulfoxide may be potential biomarkers to assess exposure and oxidative stress associated with AαC and other arylamine toxicants present in tobacco smoke.

Increasing 3D Matrix Rigidity Strengthens Proliferation and Spheroid Development of Human Liver Cells in a Constant Growth Factor Environment.

Mechanical forces influence the growth and shape of virtually all tissues and organs. Recent studies show that increased cell contractibility, growth and differentiation might be normalized by modulating cell tensions. Particularly, the role of these tensions applied by the extracellular matrix during liver fibrosis could influence the hepatocarcinogenesis process. The objective of this study is to determine if 3D stiffness could influence growth and phenotype of normal and transformed hepatocytes and to integrate extracellular matrix (ECM) stiffness to tensional homeostasis. We have developed an appropriate 3D culture model: hepatic cells within three-dimensional collagen matrices with varying rigidity. Our results demonstrate that the rigidity influenced the cell phenotype and induced spheroid clusters development whereas in soft matrices, Huh7 transformed cells were less proliferative, well-spread and flattened. We confirmed that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas ERK2 mainly controlled proliferation. As compared to 2D culture, 3D cultures are associated with epithelial markers expression. Interestingly, proliferation of normal hepatocytes was also induced in rigid gels. Furthermore, biotransformation activities are increased in 3D gels, where CYP1A2 enzyme can be highly induced/activated in primary culture of human hepatocytes embedded in the matrix. In conclusion, we demonstrated that increasing 3D rigidity could promote proliferation and spheroid developments of liver cells demonstrating that 3D collagen gels are an attractive tool for studying rigidity-dependent homeostasis of the liver cells embedded in the matrix and should be privileged for both chronic toxicological and pharmacological drug screening.

Mass Spectrometric Characterization of Human Serum Albumin Adducts Formed with N-Oxidized Metabolites of 2-Amino-1-methylphenylimidazo[4,5-b]pyridine in Human Plasma and Hepatocytes.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic heterocyclic aromatic amine formed in cooked meats, is metabolically activated to electrophilic intermediates that form covalent adducts with DNA and protein. We previously identified an adduct of PhIP formed at the Cys(34) residue of human serum albumin following reaction of albumin with the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP). The major adducted peptide recovered from a tryptic/chymotryptic digest was identified as the missed-cleavage peptide LQQC*([SO2PhIP])PFEDHVK, a [cysteine-S-yl-PhIP]-S-dioxide linked adduct. In this investigation, we have characterized the albumin adduction products of N-sulfooxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-sulfooxy-PhIP), which is thought to be a major genotoxic metabolite of PhIP formed in vivo. Targeted and data-dependent scanning methods showed that N-sulfooxy-PhIP adducted to the Cys(34) of albumin in human plasma to form LQQC*([SO2PhIP])PFEDHVK at levels that were 8-10-fold greater than the adduct levels formed with N-(acetyloxy)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP) or HONH-PhIP. We also discovered that N-sulfooxy-PhIP forms an adduct at the sole tryptophan (Trp(214)) residue of albumin in the sequence AW*([PhIP])AVAR. However, stable adducts of PhIP with albumin were not detected in human hepatocytes. Instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine (5-HO-PhIP), a solvolysis product of the proposed nitrenium ion of PhIP, were recovered during the proteolysis, suggesting a labile sulfenamide linkage had formed between an N-oxidized intermediate of PhIP and Cys(34) of albumin. A stable adduct was formed at the Tyr(411) residue of albumin in hepatocytes and identified as a deaminated product of PhIP, Y(*[desaminoPhIP])TK, where the 4-HO-tyrosine group bound to the C-2 imidazole atom of PhIP.

MEK1/2 overactivation can promote growth arrest by mediating ERK1/2-dependent phosphorylation of p70S6K.

The extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway has been involved in the positive and negative regulation of cell proliferation. Upon mitogen stimulation, ERK1/ERK2 activation is necessary for G1- to S-phase progression whereas when hyperactived, this pathway could elicit cell cycle arrest. The mechanisms involved are not fully elucidated but a kinase-independent function of ERK1/2 has been evidenced in the MAPK-induced growth arrest. Here, we show that p70S6K, a central regulator of protein biosynthesis, is essential for the cell cycle arrest induced by overactivation of ERK1/2. Indeed, whereas MEK1 silencing inhibits cell cycle progression, we demonstrate that active mutant form of MEK1 or MEK2 triggers a G1 phase arrest by stimulating an activation of p70S6K by ERK1/2 kinases. Silencing of ERK1/2 activity by shRNA efficiently suppresses p70S6K phosphorylation on Thr421/Ser424 and S6 phosphorylation on Ser240/244 as well as p21 expression, but these effects can be partially reversed by the expression of kinase-dead mutant form of ERK1 or ERK2. In addition, we demonstrate that the kinase p70S6K modulates neither the p21 gene transcription nor the stability of the protein but enhances the translation of the p21 mRNA. In conclusion, our data emphasizes the importance of the translational regulation of p21 by the MEK1/2-ERK1/2-p70S6K pathway to negatively control the cell cycle progression.

Integrative analysis of high-throughput RNAi screen data identifies the FER and CRKL tyrosine kinases as new regulators of the mitogenic ERK-dependent pathways in transformed cells.

BACKGROUND: Cell proliferation is a hallmark of cancer and depends on complex signaling networks that are chiefly supported by protein kinase activities. Therapeutic strategies have been used to target specific kinases but new methods are required to identify combined targets and improve treatment. Here, we propose a small interfering RNA genetic screen and an integrative approach to identify kinase networks involved in the proliferation of cancer cells. RESULTS: The functional siRNA screen of 714 kinases in HeLa cells identified 91 kinases implicated in the regulation of cell growth, most of them never being reported in previous whole-genome siRNA screens. Based on gene ontology annotations, we have further discriminated between two classes of kinases that, when suppressed, result in alterations of the mitotic index and provoke cell-cycle arrest. Extinguished kinases that lead to a low mitotic index mostly include kinases implicated in cytosolic signaling. In contrast, extinguished kinases that result in a high mitotic index mostly include kinases implicated in cell division. By mapping hit kinases in the PhosphPOINT phosphoprotein database, we generated scale-free networks consisting of 449 and 661 protein-protein interactions for kinases from low MI and high MI groups, respectively. Further analyses of the kinase interactomes revealed specific modules such as FER- and CRKL-containing modules that connect three members of the epidermal growth factor receptor (EGFR) family, suggesting a tight control of the mitogenic EGF-dependent pathway. Based on experimental studies, we confirm the involvement of these two kinases in the regulation of tumor cell growth. CONCLUSION: Based on a combined approach of large kinome-wide siRNA screens and ontology annotations, our study identifies for the first time two kinase groups differentially implicated in the control of cell proliferation. We further demonstrate that integrative analysis of the kinase interactome provides key information which can be used to facilitate or optimize target design for new therapeutic strategies. The complete list of protein-protein interactions from the two functional kinase groups will provide a useful database for future investigations.

Hepatotoxicity of cyanotoxin microcystin-LR in human: Insights into mechanisms of action in the 3D culture model Hepoid-HepaRG.

Microcystin-LR (MC-LR) is a potent hepatotoxin produced by harmful cyanobacterial blooms (CyanoHABs). MC-LR targets highly differentiated hepatocytes expressing organic anion transporting polypeptides OATP1B1 and OATP1B3 that are responsible for hepatocellular uptake of the toxin. The present study utilized an advanced 3D in vitro human liver model Hepoid-HepaRG based on the cultivation of collagen-matrix embedded multicellular spheroids composed of highly differentiated and polarized hepatocyte-like cells. 14-d-old Hepoid-HepaRG cultures showed increased expression of OATP1B1/1B3 and sensitivity to MC-LR cytotoxicity at concentrations >10 nM (48 h exposure, EC20 = 26 nM). MC-LR induced neither caspase 3/7 activity nor expression of the endoplasmic reticulum stress marker gene BiP/GRP78, but increased release of pro-inflammatory cytokine IL-8, indicating a necrotic type of cell death. Subcytotoxic (10 nM) and cytotoxic (≥100 nM) MC-LR concentrations disrupted hepatocyte functions, such as xenobiotic metabolism phase-I enzyme activities (cytochrome P450 1A/1B) and albumin secretion, along with reduced expression of CYP1A2 and ALB genes. MC-LR also decreased expression of HNF4A gene, a critical regulator of hepatocyte differentiation and function. Genes encoding hepatobiliary membrane transporters (OATP1B1, BSEP, NTCP), hepatocyte gap junctional gene connexin 32 and the epithelial cell marker E-cadherin were also downregulated. Simultaneous upregulation of connexin 43 gene, primarily expressed by liver progenitor and non-parenchymal cells, indicated a disruption of tissue homeostasis. This was associated with a shift in the expression ratio of E-cadherin to N-cadherin towards the mesenchymal cell marker, a process linked to epithelial-mesenchymal transition (EMT) and hepatocarcinogenesis. The effects observed in the human liver cell in vitro model revealed mechanisms that can potentially contribute to the MC-LR-induced promotion and progression of hepatocellular carcinoma (HCC). Hepoid-HepaRG cultures provide a robust, accessible and versatile in vitro model, capable of sensitively detecting hepatotoxic effects at toxicologically relevant concentrations, allowing for assessing hepatotoxicity mechanisms, human health hazards and impacts of environmental hepatotoxins, such as MC-LR.

Assessment of endocrine disruptor impacts on lipid metabolism in a fatty acid-supplemented HepaRG human hepatic cell line.

The incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing worldwide. This disease encompasses several stages, from steatosis to steatohepatitis and, eventually, to fibrosis and cirrhosis. Exposure to environmental contaminants is one of the risk factors and an increasing amount of evidence points to a role for endocrine disrupting compounds (EDCs). This study assesses the impact of selected EDCs on the formation of lipid droplets, the marker for steatosis in a hepatic model. The mechanisms underlying this effect are then explored. Ten compounds were selected according to their obesogenic properties: bisphenol A, F and S, butyl-paraben, cadmium chloride, p,p'-DDE, DBP, DEHP, PFOA and PFOS. Using a 2D or 3D model, HepaRG cells were exposed to the compounds with or without fatty acid supplementation. Then, the formation of lipid droplets was quantified by an automated fluorescence-based method. The expression of genes and proteins involved in lipid metabolism and the impact on cellular respiration was analyzed. The formation of lipid droplets, which is revealed or enhanced by oleic acid supplementation, was most effectively induced by p,p'-DDE and DEHP. Experiments employing either 2D or 3D culture conditions gave similar results. Both compounds induced the expression of PLIN2. p,p'-DDE also appears to act by decreasing in fatty acid oxidation. Some EDCs were able to induce the formation of lipid droplets, in HepaRG cells, an effect which was increased after supplementation of the cells with oleic acid. A full understanding of the mechanisms of these effects will require further investigation. The novel automated detection method described here may also be useful in the future as a regulatory test for EDC risk assessment.

MAPK signaling in cisplatin-induced death: predominant role of ERK1 over ERK2 in human hepatocellular carcinoma cells.

Hepatocellular carcinoma treatment by arterial infusion of cis-diamminedichloroplatinum-II (cisplatin) exhibits certain therapeutic efficacy. However, optimizations are required and the mechanisms underlying cisplatin proapoptotic effect remain unclear. The mitogen-activated protein kinase (MAPK) pathway plays a key role in cell response to cisplatin and the functional specificity of the isoform MAPK/ERK kinase 1 and 2 (MEK1/2) and ERK1/2 could influence this response. The individual contribution of each kinase on cisplatin-induced death was thus analyzed after a transient or stable specific inhibition by RNA interference in the human hepatocellular carcinoma cells Huh-7 or in knockout mice. We demonstrated here that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas MEK1 and MEK2 acted in a redundant manner. Indeed, at clinically relevant concentrations of cisplatin, ERK1 silencing alone was sufficient to protect cells from cisplatin-induced death both in vitro, in Huh-7 cells and ERK1(-/-) hepatocytes, and in vivo, in ERK1-deficient mice. Moreover, we showed that ERK1 activity correlated with the induction level of the proapoptotic BH3-only protein Noxa, a critical mediator of cisplatin toxicity. On the contrary, ERK2 inhibition upregulated ERK1 activity, favored Noxa induction and sensitized hepatocarcinoma cells to cisplatin. Our results point to a crucial role of ERK1 in cisplatin-induced proapoptotic signal and lead us to propose that ERK2-specific targeting could improve the efficacy of cisplatin therapy by increasing ERK1 prodeath functions.

UDP-glucuronosyltransferase-mediated metabolic activation of the tobacco carcinogen 2-amino-9H-pyrido[2,3-b]indole.

2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of AαC and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by (1)H NMR and mass spectrometry. AαC and HONH-AαC underwent glucuronidation by UGTs to form, respectively, N(2)-(ß-D-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N(2)-Gl) and N(2)-(ß-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HON(2)-Gl). HONH-AαC also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(ß-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN(2)-O-Gl). AαC-HN(2)-O-Gl is a biologically reactive metabolite and binds to calf thymus DNA (pH 5.0 or 7.0) to form the N-(deoxyguanosin-8-yl)-AαC adduct at 20-50-fold higher levels than the adduct levels formed with HONH-AαC. Major UGT isoforms were examined for their capacity to metabolize AαC and HONH-AαC. UGT1A4 was the most catalytically efficient enzyme (V(max)/K(m)) at forming AαC-N(2)-Gl (0.67 µl·min(-1)·mg of protein(-1)), and UGT1A9 was most catalytically efficient at forming AαC-HN-O-Gl (77.1 µl·min(-1)·mg of protein(-1)), whereas UGT1A1 was most efficient at forming AαC-HON(2)-Gl (5.0 µl·min(-1)·mg of protein(-1)). Human hepatocytes produced AαC-N(2)-Gl and AαC-HN(2)-O-Gl in abundant quantities, but AαC-HON(2)-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-AαC.

DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ∼5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a 10,000-fold concentration range (1 nM-10 µM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes suggest that AαC can contribute to DNA damage and the risk of hepatocellular cancer in smokers.

ReadEDTest: A tool to assess the readiness of in vitro test methods under development for identifying endocrine disruptors.

Growing evidence shows that endocrine disruptors (EDs), known to affect the reproductive system, may also disturb other hormone-regulated functions leading to cancers, neurodevelopmental defects, metabolic and immune diseases. To reduce exposure to EDs and limit their health effects, development of screening and mechanism-based assays to identify EDs is encouraged. Nevertheless, the crucial validation step of test methods by regulatory bodies is a time- and resource-consuming process. One of the main raisons of this long duration process is that method developers, mainly researchers, are not fully aware of the regulatory needs to validate a test. We propose an online self-assessment questionnaire (SAQ) called ReadEDTest easy to be used by all researchers. The aim of ReadEDTest is to speed up the validation process by assessing readiness criteria of in vitro and fish embryo ED test methods under development. The SAQ is divided into 7 sections and 13 sub-sections containing essential information requested by the validating bodies. The readiness of the tests can be assessed by specific score limits for each sub-section. Results are displayed via a graphical representation to help identification of the sub-sections having sufficient or insufficient information. The relevance of the proposed innovative tool was supported using two test methods already validated by the OECD and four under development test methods.

DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 4-aminobiphenyl are infrequently detected in human mammary tissue by liquid chromatography/tandem mass spectrometry.

Some epidemiological investigations have revealed that frequent consumption of well-done cooked meats and tobacco smoking are risk factors for breast cancer in women. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed in well-done cooked meat, and 4-aminobiphenyl (4-ABP) is an aromatic amine that arises in tobacco smoke and occurs as a contaminant in the atmosphere. Both compounds are rodent mammary carcinogens, and putative DNA adducts of PhIP and 4-ABP have been frequently detected, by immunohistochemistry (IHC) or (32)P-post-labeling methods, in mammary tissue of USA women. Because of these findings, PhIP and 4-ABP have been implicated as causal agents of human breast cancer. However, the biomarker data are controversial: both IHC and (32)P-post-labeling are non-selective screening methods and fail to provide confirmatory spectral data. Consequently, the identities of the lesions are equivocal. We employed a specific and sensitive liquid chromatography/mass spectrometry (MS) method, to screen tumor-adjacent normal mammary tissue for DNA adducts of PhIP and 4-ABP. Only 1 of 70 biopsy samples obtained from Minneapolis, Minnesota breast cancer patients contained a PhIP-DNA adduct. The level was three adducts per 10(9) nucleotides, a level that is 100-fold lower than the mean level of PhIP adducts reported by IHC or (32)P-post-labeling methods. The occurrence of 4-ABP-DNA adducts was nil in those same breast tissues. Our findings, derived from a specific mass spectrometry method, signify that PhIP and 4-ABP are not major DNA-damaging agents in mammary tissue of USA women and raise questions about the roles of these chemicals in breast cancer.

The complexity of ERK1 and ERK2 MAPKs in multiple hepatocyte fate responses.

Recent reports suggest that extracellular signal-regulated kinase (ERK1) and ERK2 mitogen-activated protein kinases (MAPK) may direct specific biological functions under certain contexts. In this study, we investigated the role of early and sustained epidermal growth factor (EGF) stimulation on long-term hepatocyte differentiation and the possible role of ERK1 and ERK2 in this process. We demonstrate a long-term survival and an elevated level of differentiation up to 3 weeks. The differentiation state of hepatocytes is supported by sustained expression of aldolase B, albumin, and the detoxifying enzymes CYP1A2, 2B2, and 3A23. Similarly to freshly isolated cells, cultured hepatocytes also retain the ability to respond to 3-methylcholanthrene (3MC) and phenobarbital (PB), two known CYP inducers. In addition, we show evidence that continuous MAPK/ERK kinase (MEK) inhibition enhances the level of differentiation. Using RNA interference approaches against ERK1 and ERK2, we demonstrate that this effect requires both ERK1 and ERK2 activity, whereas the specific ERK1 knockdown promotes cell survival and the specific ERK2 knockdown regulates cell proliferation. In conclusion, we demonstrate that early and sustained EGF stimulation greatly extends long-term hepatocyte survival and differentiation, and that inhibition of the ERK1/2 MAPK pathway potentiates these pro-survival/pro-differentiation phenotypes. We clearly attest that specific ERK1 and ERK2 MAPKs determine hepatocyte survival and proliferation, respectively, whereas dual inhibition is required to stabilize a highly differentiated state.

In vitrolong term differentiation and functionality of three-dimensional bioprinted primary human hepatocytes: application forin vivoengraftment.

In recent decades, 3Din vitrocultures of primary human hepatocytes (PHHs) have been increasingly developed to establish models capable of faithfully mimicking main liver functions. The use of 3D bioprinting, capable of recreating structures composed of cells embedded in matrix with controlled microarchitectures, is an emergent key feature for tissue engineering. In this work, we used an extrusion-based system to print PHH in a methacrylated gelatin (GelMa) matrix. PHH bioprinted in GelMa rapidly organized into polarized hollow spheroids and were viable for at least 28 d of culture. These PHH were highly differentiated with maintenance of liver differentiation genes over time, as demonstrated by transcriptomic analysis and functional approaches. The cells were polarized with localization of apico/canalicular regions, and displayed activities of phase I and II biotransformation enzymes that could be regulated by inducers. Furthermore, the implantation of the bioprinted structures in mice demonstrated their capability to vascularize, and their ability to maintain human hepatic specific functions for at least 28 d was illustrated by albumin secretion and debrisoquine metabolism. This model could hold great promise for human liver tissue generation and its use in future biotechnological developments.

Obesity III: Obesogen assays: Limitations, strengths, and new directions.

There is increasing evidence of a role for environmental contaminants in disrupting metabolic health in both humans and animals. Despite a growing need for well-understood models for evaluating adipogenic and potential obesogenic contaminants, there has been a reliance on decades-old in vitro models that have not been appropriately managed by cell line providers. There has been a quick rise in available in vitro models in the last ten years, including commercial availability of human mesenchymal stem cell and preadipocyte models; these models require more comprehensive validation but demonstrate real promise in improved translation to human metabolic health. There is also progress in developing three-dimensional and co-culture techniques that allow for the interrogation of a more physiologically relevant state. While diverse rodent models exist for evaluating putative obesogenic and/or adipogenic chemicals in a physiologically relevant context, increasing capabilities have been identified for alternative model organisms such as Drosophila, C. elegans, zebrafish, and medaka in metabolic health testing. These models have several appreciable advantages, including most notably their size, rapid development, large brood sizes, and ease of high-resolution lipid accumulation imaging throughout the organisms. They are anticipated to expand the capabilities of metabolic health research, particularly when coupled with emerging obesogen evaluation techniques as described herein.

Combinatorial pathway disruption is a powerful approach to delineate metabolic impacts of endocrine disruptors.

The prevalence of metabolic diseases, such as obesity, diabetes, metabolic syndrome and chronic liver diseases among others, has been rising for several years. Epidemiology and mechanistic (in vivo, in vitro and in silico) toxicology have recently provided compelling evidence implicating the chemical environment in the pathogenesis of these diseases. In this review, we will describe the biological processes that contribute to the development of metabolic diseases targeted by metabolic disruptors, and will propose an integrated pathophysiological vision of their effects on several organs. With regard to these pathomechanisms, we will discuss the needs, and the stakes of evolving the testing and assessment of endocrine disruptors to improve the prevention and management of metabolic diseases that have become a global epidemic since the end of last century.

Obesity II: Establishing causal links between chemical exposures and obesity.

Obesity is a multifactorial disease with both genetic and environmental components. The prevailing view is that obesity results from an imbalance between energy intake and expenditure caused by overeating and insufficient exercise. We describe another environmental element that can alter the balance between energy intake and energy expenditure: obesogens. Obesogens are a subset of environmental chemicals that act as endocrine disruptors affecting metabolic endpoints. The obesogen hypothesis posits that exposure to endocrine disruptors and other chemicals can alter the development and function of the adipose tissue, liver, pancreas, gastrointestinal tract, and brain, thus changing the set point for control of metabolism. Obesogens can determine how much food is needed to maintain homeostasis and thereby increase the susceptibility to obesity. The most sensitive time for obesogen action is in utero and early childhood, in part via epigenetic programming that can be transmitted to future generations. This review explores the evidence supporting the obesogen hypothesis and highlights knowledge gaps that have prevented widespread acceptance as a contributor to the obesity pandemic. Critically, the obesogen hypothesis changes the narrative from curing obesity to preventing obesity.