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

International audienceMechanical 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

Metabolic activation and genotoxicity of Heterocyclic Amines Aromatics (AHA) in humans

Les amines hétérocycliques aromatiques (AHA) sont des contaminants de l'environnement et de l'alimentation, majoritairement formés lors de la cuisson de viande et poisson ainsi que dans la fumée de cigarette et les gaz d'échappements. Les AHA sont mutagènes chez la bactérie, cancérogènes multi-sites chez le rongeur et sont classées comme cancérogènes possibles ou probables chez l'Homme par l'IARC. Il est aujourd'hui indispensable de caractériser des biomarqueurs d'exposition dérivés des AHA (adduits à l'ADN et métabolites) pour améliorer l'estimation du risque chez l'Homme. Des résultats de l'équipe ont démontré que le 2-amino-9H-pyrido[2,3-b]indole (AαC) forme des niveaux d'adduits à l'ADN élevés dans les hépatocytes humains. Ces niveaux sont plus élevés que ceux formés par les autres AHA. L'objectif de cette thèse est de mieux comprendre le potentiel génotoxique d'AαC chez l'Homme. Nos travaux ont démontré que les adduits à l'ADN dérivés d'AαC sont persistants dans les hépatocytes humains et formés à des doses aussi faibles que 1nM. De plus, le CYP1A2 a été confirmé comme enzyme majoritaire dans la bioactivation d'AαC dans le foie humain. Nous avons également caractérisé les métabolites majeurs dérivés d'AαC dans les hépatocytes humains. Cette étude a permis d'établir pour la première fois une corrélation entre l'activité catalytique du CYP1A2, la formation d'AαC-HN2-O-Gl et la formation des adduits à l'ADN dérivés d'AαC. Le métabolite AαC-HN2-O-Gl étant réactif vis-à-vis de l'ADN in vitro, nos travaux confortent l'hypothèse que la voie des UDP-Glucuronosyltransférases (UGTs) est une nouvelle voie de bioactivation d'AαC dans le foie humain. De plus, nous avons montré que les adduits à l'ADN dérivés des AHA sont formés dans les lymphocytes T humains activés et en particulier les adduits en position C8 de la guanine dérivés d'AαC. Au total, ces travaux ont permis l'identification de métabolites stables et des adduits à l'ADN, potentiels biomarqueurs d'exposition à AαC, qui sont indispensables pour une meilleure estimation du risque génotoxique d'AαC chez l'Homme.Heterocyclic aromatic amines (HAA) are environmental and food contaminants, mainly formed during meat and fish cooking, but also in cigarette smoke and exhaust gaz. HAA are mutagenic in bacteria, carcinogenic in rodents and are classified as possible or probable human carcinogens by IARC. Today it is essential to characterize exposure biomarkers i.e. DNA adducts and metabolites, to assess the human risk associated with HAA. The research team has previously demonstrated that 2-amino-9H-pyrido[2,3-b]indole (AαC) form high levels of DNA adducts in human hepatocytes. These levels are greater that those derived from other HAAs. Thus, the aim of this thesis was to better understand the genotoxic potential of AαC in human. We demonstrated that in human hepatocytes, DNA adducts derived from AαC are persistent and formed at doses as low as 1nM. Moreover, we confirmed that CYP1A2 is the major enzyme implicated in the bioactivation of AαC in human liver. We have also characterized the major metabolites derived from AαC formed in human hepatocytes. This study allows, for the first time, the establishment of a correlation between the catalytic activity of CYP1A2, AαC-HN2-O-Gl formation and AαC derived DNA adducts formation. AαC-HN2-O-Gl being reactive toward DNA in vitro, our work reinforces the hypothesis that the UDP-glucuronosyltransferase (UGTs) pathway is a new bioactivation pathway for AαC in human liver. Moreover, we demonstrated the formation of HAA derived DNA adducts, especially those derived from AαC at position C8 of guanine, in activated human T lymphocytes. Taken together, our data lead to the identification of stable metabolites as well as DNA adducts which are potentials AαC exposure biomarkers in human. These biomarkers are essential for a better assessment of the genotoxic risk of AαC in human

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

International audienceno abstrac

THE HETEROCYCLIC AROMATIC AMINE, 2-AMINO-3-METHYLIMIDAZO [4,5-F] QUINOLINE (IQ) INDUCES HUMAN CYTOCHROME P450 1A2 THROUGH THE ARYL HYDROCARBON RECEPTOR (AHR) AND XENOBIOTIC RESPONSIVE ELEMENT (XRE) IN HUMAN HEPATOCYTES

International audienceMeeting abstract: 19th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX) / 29th Meeting of the Japanese-Society-for-the-Study-of-Xenobiotics (JSSX

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

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.

International audience2-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 Cys34 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 Cys34 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 (Trp214) 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 Cys34 of albumin. A stable adduct was formed at the Tyr411 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

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

International audience2-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 smok

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.

International audienceAromatic 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

Metabolism of the Tobacco Carcinogen 2‑Amino‑9<i>H</i>‑pyrido[2,3‑<i>b</i>]indole (AαC) in Primary Human Hepatocytes

2-Amino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­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, <i>N</i>²-acetyl-2-amino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indole-6-yl-oxo-(β-d-glucuronic acid) (<i>N</i>²-acetyl-AαC-6-<i>O</i>-Gluc); two ring-oxidized glucuronide (Gluc) conjugates: 2-amino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indol-3-yl-oxo-(β-d-glucuronic acid) (AαC-3-<i>O</i>-Gluc) and 2-amino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indol-6-yl-oxo-(β-d-glucuronic acid) (AαC-6-<i>O</i>-Gluc); two sulfate conjugates, 2-amino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indol-3-yl sulfate (AαC-3-<i>O</i>-SO₃H) and 2-amino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indol-6-yl sulfate (AαC-6<i>-O-</i>SO₃H); and the Gluc conjugate, <i>N</i>²-(β-d-glucosidurony1)-2-amino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indole (AαC-<i>N</i>²-Gluc). In addition, four minor metabolites were identified: <i>N</i>²-acetyl-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indol-3-yl sulfate (<i>N</i>²-acetyl-AαC-3-<i>O</i>-SO₃H), <i>N</i>²-acetyl-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indol-6-yl sulfate (<i>N</i>²-acetyl-AαC-6-<i>O</i>-SO₃H), <i>N</i>²-acetyl-2-amino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indol-3-yl-oxo-(β-d-glucuronic acid) (<i>N</i>²-acetyl-AαC-3<i>-O</i>-Gluc), and <i>O</i>-(β-d-glucosidurony1)-2-hydroxyamino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­indole (AαC-HN²-<i>O</i>-Gluc). The latter metabolite, AαC-HN²-<i>O</i>-Gluc is a reactive intermediate that binds to DNA to form the covalent adduct <i>N</i>-(2′-deoxyguanosin-8-yl)-2-amino-9<i>H</i>-pyrido­[2,3-<i>b</i>]­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-<i>O</i>-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-<i>O</i>-Gluc conjugate and AαC-DNA adducts

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

2-Amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­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 Cys³⁴ residue of human serum albumin following reaction of albumin with the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­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 <i>N</i>-sulfooxy-2-amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (<i>N</i>-sulfooxy-PhIP), which is thought to be a major genotoxic metabolite of PhIP formed <i>in vivo</i>. Targeted and data-dependent scanning methods showed that <i>N</i>-sulfooxy-PhIP adducted to the Cys³⁴ of albumin in human plasma to form LQ­QC*^[SO₂PhIP]PF­E­D­H­VK at levels that were 8–10-fold greater than the adduct levels formed with <i>N</i>-(acetyloxy)-2-amino-1-methyl-6-phenylimidazo­[4,5-<i>b</i>]­pyridine (<i>N</i>-acetoxy-PhIP) or HONH-PhIP. We also discovered that <i>N</i>-sulfooxy-PhIP forms an adduct at the sole tryptophan (Trp²¹⁴) 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-<i>b</i>]­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 Cys³⁴ of albumin. A stable adduct was formed at the Tyr⁴¹¹ 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