Éditeur(s) : HAL CCSD American Chemical Society
Résumé : International audience
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.
ISSN: 0893-228X

inserm-00869907
http://www.hal.inserm.fr/inserm-00869907
DOI : 10.1021/tx4002226
PUBMED : 23898916

Éditeur(s) : HAL CCSD
Résumé : International audience
STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase₉) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.
Molecular Human Reproduction

hal-01647081
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01647081
DOI : 10.1093/molehr/gax054
PUBMED : 29040674

Éditeur(s) : HAL CCSD Federation of American Society of Experimental Biology
Résumé : International audience
Testes of most male mammals present the particularity of being externalized from the body and are consequently slightly cooler than core body temperature (4-8 degrees C below). Although, hypothermia of the testis is known to increase germ cells apoptosis, little is known about the underlying molecular mechanisms, including cold sensors, transduction pathways, and apoptosis triggers. In this study, using a functional knockout mouse model of the cold and menthol receptors, dubbed transient receptor potential melastatine 8 (TRPM8) channels, we found that TRPM8 initiated the cold-shock response by differentially modulating cold- and heat-shock proteins. Besides, apoptosis of germ cells increased in proportion to the cooling level in control mice but was independent of temperature in knockout mice. We also observed that the rate of germ cell death correlated positively with the reactive oxygen species level and negatively with the expression of the detoxifying enzymes. This result suggests that the TRPM8 sensor is a key determinant of germ cell fate under hypothermic stimulation.Borowiec, A.-S., Sion, B., Chalmel, F., Rolland, A. D., Lemonnier, L., De Clerck, T., Bokhobza, A., Derouiche, S., Dewailly, E., Slomianny, C., Mauduit, C., Benahmed, M., Roudbaraki, M., Jegou, B., Prevarskaya, N., Bidaux, G. Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.
ISSN: 0892-6638

hal-01390973
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390973
DOI : 10.1096/fj.201600257R
PUBMED : 27317670

Éditeur(s) : Université de Bretagne Occidentale
Résumé : Archaea are micro-organisms present in all ecosystems, but are majoritary present in extremophilic environments. Hyperthermophilic Archaea, as Pyrococcus abyssi, are permanently exposed at temperature which can increase the rate of spontaneous DNA damage. It is probable that hyperthermophilic Archaea possess efficient molecular mechanism to duplicate, maintain and stabilize their genomes. The aim of this project was to investigate the interaction network involved in the process of DNA replication and DNA repair. The methodological approach consists in the coupling of pull-down with mass spectrometry identification of interacting partners. A preliminary interaction network was identified that was composed of new proteins as well as unsuspected interactions between known components of DNA replication machinery. The main results consist in: (1) the nuclease Pab2263, which interacts with PCNA, is a novel player in DNA metabolism. (2) PCNA is likely part of a macrocomplex with the ubiquitary proteins Mre11 and Rad50 suggesting a role of this complex in the repair of double strand break in connection with a replication fork. (3) Fen1 and DNA primase interact physically and can collaborate in vitro to resolve intermediate step of the base excision repair pathway. These results enhance our understanding of DNA repair process in Archaea.
Les Archaea sont des micro-organismes rencontrés dans tous les écosystèmes, mais qui apparaissent comme majoritaires dans les environnements dits extrêmes. Les archaea hyperthermophiles, comme Pyrococcus abyssi sont en permanence exposées à des températures qui peuvent augmenter le taux de dommages de l'ADN, pourtant, le taux de mutations spontanés chez ces micro-organismes est similaire à celui des espèces modèles mésophiles. Il est ainsi probable que les hyperthermophiles possèdent des systèmes particulièrement efficaces pour dupliquer, maintenir et stabiliser leur génome. L'objectif de ce projet était d'explorer le réseau d'interaction impliqué dans les processus de réplication et de réparation de l'ADN. L'approche méthodologique mise en oeuvre a consisté à coupler la capture de partenaires d'interaction par pull-down avec leur identification par spectrométrie de masse. J'ai pu ainsi mettre en évidence, au sein l'extrait cellulaire de P. abyssi, un réseau préliminaire reliant des protéines de la maintenance génomique. Nous avons non seulement mis en évidence de nouvelles protéines impliquées probablement dans des mécanismes de réparation, mais également des nouvelles interactions non suspectées entre des composants déjà caractérisés. Les principaux résultats sont les suivants : (1) La nucléase Pab2263, partenaire du PCNA, est un nouvel acteur du métabolisme de l'ADN. (2) Le PCNA forme un macrocomplexe avec les protéines ubiquitaires Mre11 et Rad50 suggérant un rôle de ce complexe dans la réparation des cassures double brin de l'ADN lors de la réplication. (3) Les protéines Fen1 et l'ADN primase interagissent physiquement et peuvent collaborer in vitro pour résoudre une étape intermédiaire de la voie de réparation par excision de base. Ces résultats enrichissent notre compréhension des processus de réparation de l'ADN chez les archées.

Droits : info:eu-repo/semantics/openAccess
http://archimer.ifremer.fr/doc/2008/these-3722.pdf
http://archimer.ifremer.fr/doc/00000/3722/

Éditeur(s) : HAL CCSD American Association for Cancer Research
Résumé : International audience
The aim of this study was to test in vitro the efficacy of TAC, an original G-quadruplex ligand, as a potential radiosensitizing agent for glioblastoma multiforme (GBM). Two human radioresistant telomerase-positive GBM cell lines (SF763 and SF767) were analyzed, with and without TAC treatment, for telomere length, cell proliferation, apoptosis, cell-cycle distribution, gene expression, cytogenetic aberrations, clonogenic survival assay, 53BP1 immunofluorescence staining, and γH2AX phosphorylation. We found that low concentrations of TAC (0.5 and 1 μmol/L) inhibited the proliferation of GBM cells in a concentration-dependent manner after only 1 week of treatment, with minimal effects on cell cycle and apoptosis. TAC treatment had no visible effect on average telomere length but modified expression levels of telomere-related genes (hTERT, TRF1, and TRF2) and induced concentration-dependent DNA damage response and dicentric chromosomes. Survival curves analysis showed that exposure to nontoxic, subapoptotic concentrations of TAC enhanced radiation-induced killing of GBM cells. Analysis of DNA repair after irradiation revealed delayed repair kinetics in GBM cells treated with TAC. Furthermore, the combined treatment (TAC and radiation) significantly increased the frequency of chromosomal aberrations as compared with radiation alone. These findings provide the first evidence that exposure to a G4 ligand radiosensitizes human glioblastoma cells and suggest the prospect of future therapeutic applications.
ISSN: 1535-7163

hal-00851184
https://hal.archives-ouvertes.fr/hal-00851184
DOI : 10.1158/1535-7163.MCT-10-0664
PUBMED : 21987532

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are widely distributed environmental contaminants exerting toxic effects such as genotoxicity and carcinogenicity, mainly associated with aryl hydrocarbon receptor (AhR) activation and the subsequent induction of cytochromes P-450 (CYP) 1-metabolizing enzymes. We previously reported an up-regulation of AhR expression and activity in primary cultures of human T lymphocyte by a physiological activation. Despite the suggested link between exposure to PAHs and the risk of lymphoma, the potential of activated human T lymphocytes to metabolize AhR exogenous ligands such as B[a]P, and produce DNA damage has not been investigated. In the present study, we characterized the genotoxic response of primary activated T lymphocytes to B[a] P. We demonstrated that, following T lymphocyte activation, B[a]P treatment triggers a marked increase in CYP1 expression and activity generating, upon metabolic activation, DNA adducts and double-strand breaks (DSBs) after a 48-h treatment. At this time point, B[a]P also induces a DNA damage response with ataxia telangiectasia mutated kinase activation, thus producing a p53-dependent response and T lymphocyte survival. B[a]P activates DSB repair by mobilizing homologous recombination machinery but also induces gene mutations in activated human T lymphocytes which could consequently drive a cancer process. In conclusion, primary cultures of activated human T lymphocytes represent a good model for studying genotoxic effects of environmental contaminants such as PAHs, and predicting human health issues.
ISSN: 0006-2952

hal-01560145
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01560145
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01560145/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01560145/file/Liamin%20et%20al.%20-%20Benzo%5Ba%5Dpyrene-induced%20DNA%20damage%20associated%20with%20.pdf
DOI : 10.1016/j.bcp.2017.04.025

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
The mycotoxin alternariol (AOH), a frequent contaminant in fruit and grain, is known to induce cellular stress responses such as reactive oxygen production, DNA damage and cell cycle arrest. Cellular stress is often connected to autophagy, and we employed the RAW264.7 macrophage model to test the hypothesis that AOH induces autophagy. Indeed, AOH treatment led to a massive increase in acidic vacuoles often observed upon autophagy induction. Moreover, expression of the autophagy marker LC3 was markedly increased and there was a strong accumulation of LC3-positive puncta. Increased autophagic activity was verified biochemically by measuring the degradation rate of long-lived proteins. Furthermore, AOH induced expression of Sestrin2 and phosphorylation of AMPK as well as reduced phosphorylation of mTOR and S6 kinase, common mediators of signaling pathways involved in autophagy. Transmission electron microscopy analyzes of AOH treated cells not only clearly displayed structures associated with autophagy such as autophagosomes and autolysosomes, but also the appearance of lamellar bodies. Prolonged AOH treatment resulted in changed cell morphology from round into more star-shaped as well as increased β-galactosidase activity. This suggests that the cells eventually entered senescence. In conclusion, our data identify here AOH as an inducer of both autophagy and senescence. These effects are suggested to be to be linked to AOH-induced DSB (via a reported effect on topoisomerase activity), resulting in an activation of p53 and the Sestrin2-AMPK-mTOR-S6K signaling pathway.
ISSN: 0300-483X

hal-01117627
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01117627
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01117627/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01117627/file/Autophagy%20and%20senescence_accepted.pdf
PUBMED : 25456271

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Plasma membrane is an early target of polycyclic aromatic hydrocarbons (PAH). We previously showed that the PAH prototype, benzo[a]pyrene (B[a]P), triggers apoptosis via DNA damage-induced p53 activation (genotoxic pathway) and via remodeling of the membrane cholesterol-rich microdomains called lipid rafts, leading to changes in pH homeostasis (non-genotoxic pathway). As omega-3 (n-3) fatty acids can affect membrane composition and function or hamper in vivo PAH genotoxicity, we hypothesized that addition of physiologically relevant levels of polyunsaturated n-3 fatty acids (PUFAs) might interfere with B[a]P-induced toxicity. The effects of two major PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), were tested on B[a]P cytotoxicity in the liver epithelial cell line F258. Both PUFAs reduced B[a]P-induced apoptosis. Surprisingly, pre-treatment with DHA increased the formation of reactive B[a]P metabolites, resulting in higher levels of B[a]P-DNA adducts. EPA had no apparent effect on B[a]P metabolism or related DNA damage. EPA and DHA prevented B[a]P-induced apoptotic alkalinization by affecting Na(+)/H(+) exchanger 1 activity. Thus, the inhibitory effects of omega-3 fatty acids on B[a]P-induced apoptosis involve a non-genotoxic pathway associated with plasma membrane remodeling. Our results suggest that dietary omega-3 fatty acids may have marked effects on the biological consequences of PAH exposure.
ISSN: 0009-2797

hal-00950341
https://hal.archives-ouvertes.fr/hal-00950341
https://hal.archives-ouvertes.fr/hal-00950341/document
https://hal.archives-ouvertes.fr/hal-00950341/file/DENDELE_et_al_2013-unmarked.pdf
PUBMED : 24246761

Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
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.
ISSN: 0143-3334

hal-00682453
https://hal.archives-ouvertes.fr/hal-00682453
DOI : 10.1093/carcin/bgr252
PUBMED : 22072616

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Background & Aims: The severity of liver diseases is exacerbated by the death of hepatocytes, which can be induced by the sensing of pathogen associated molecular patterns (PAMPs) derived from the gut microbiota. The molecular mechanisms regulating these cell death pathways are poorly documented. In this study, we investigated the role of the receptor interacting protein kinase 1 (RIPK1), a protein known to regulate cell fate decisions, in the death of hepatocytes using two in vivo models of PAMP-induced hepatitis. Methods: Hepatitis was induced in mice by independent injections of two different bacterial PAMPs: lipopolysaccharide (LPS) and unmethylated CpG oligodeoxynucleotide (CpG-DNA) motifs. The role of RIPK1 was evaluated by using mice specifically lacking RIPK1 in liver parenchymal cells (Ripk1(LPC-KO)). Administration of liposome-encapsulated clodronate served to investigate the role of Kupffer cells in the establishment of the disease. Etanercept, a tumor necrosis factor (TNF)-decoy receptor, was used to study the contribution of TNF-alpha during LPS-mediated liver injury. Results: Whereas RIPK1 deficiency in liver parenchymal cells did not trigger basal hepatolysis, it greatly sensitized hepatocytes to apoptosis and liver damage following a single injection of LPS or CpG-DNA. Importantly, hepatocyte death was prevented by previous macrophage depletion or by TNF inhibition. Conclusions: Our data highlight the pivotal function of RIPK1 in maintaining liver homeostasis in conditions of macrophage-induced TNF burst in response to PAMPs sensing. Lay summary: Excessive death of hepatocytes is a characteristic of liver injury. A new programmed cell death pathway has been described involving upstream death ligands such as TNF and downstream kinases such as RIPK1. Here, we show that in the presence of LPS liver induced hepatic injury was due to secretion of TNF by liver macrophages, and that RIPK1 acts as a powerful protector of hepatocyte death. This newly identified pathway in the liver may be helpful in the management of patients to predict their risk of developing acute liver failure. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
ISSN: 0168-8278

hal-01532060
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01532060
DOI : 10.1016/j.jhep.2017.01.005
PUBMED : 28088582

Éditeur(s) : HAL CCSD American Society for Biochemistry and Molecular Biology
Résumé : International audience
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
ISSN: 0021-9258

hal-01169796
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01169796
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01169796/file/Manuscript_KP_JBC_REV_050515_final_TC.pdf
DOI : 10.1074/jbc.M115.646539
PUBMED : 25953894

Éditeur(s) : HAL CCSD
Résumé : International audience
We used K562 cells sensitive or generated resistant to imatinib or nilotinib to investigate their response to mycophenolic acid (MPA). MPA induced DNA damage leading to cell death with a minor contribution of apoptosis, as revealed by annexin V labeling (up to 25%). In contrast, cell cycle arrest and positive staining for senescence-associated β-galactosidase activity were detected for a large cell population (80%). MPA-induced cell death was potentialized by the inhibition of autophagy and this is associated to the upregulation of apoptosis. In contrast, senescence was neither decreased nor abrogated in autophagy deficient K562 cells. Primary CD34 cells from CML patients sensitive or resistant to imatinib or nilotinib respond to MPA although apoptosis is mainly detected. These results show that MPA is an interesting tool to overcome resistance in vitro and in vivo mainly in the evolved phase of the disease.
Leukemia Research and Treatment

hal-00846103
https://hal.archives-ouvertes.fr/hal-00846103
https://hal.archives-ouvertes.fr/hal-00846103/document
https://hal.archives-ouvertes.fr/hal-00846103/file/Mycophenolic_Acid_Overcomes-accepted.pdf
PUBMED : 23213550

Éditeur(s) : HAL CCSD American Chemical Society
Résumé : International audience
The HIWATE (Health Impacts of long-term exposure to disinfection byproducts in drinking WATEr) project was a systematic analysis that combined the epidemiology on adverse pregnancy outcomes and other health effects with long-term exposure to low levels of drinking water disinfection byproducts (DBPs) in the European Union. The present study focused on the relationship of the occurrence and concentration of DBPs with in vitro mammalian cell toxicity. Eleven drinking water samples were collected from five European countries. Each sampling location corresponded with an epidemiological study for the HIWATE program. Over 90 DBPs were identified; the range in the number of DBPs and their levels reflected the diverse collection sites, different disinfection processes, and the different characteristics of the source waters. For each sampling site, chronic mammalian cell cytotoxicity correlated highly with the numbers of DBPs identified and the levels of DBP chemical classes. Although there was a clear difference in the genotoxic responses among the drinking waters, these data did not correlate as well with the chemical analyses. Thus, the agents responsible for the genomic DNA damage observed in the HIWATE samples may be due to unresolved associations of combinations of identified DBPs, unknown emerging DBPs that were not identified, or other toxic water contaminants. This study represents the first to integrate quantitative in vitro toxicological data with analytical chemistry and human epidemiologic outcomes for drinking water DBPs.
ISSN: 0013-936X

hal-00875923
https://hal.archives-ouvertes.fr/hal-00875923
DOI : 10.1021/es3024226
PUBMED : 22958121

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
The mycotoxin alternariol (AOH), a frequent contaminant in fruit and cereal products, is known to induce DNA damage with subsequent cell cycle arrest. Here we elucidated the effects of AOH on stages of cell cycle progression using the RAW 264.7 macrophage model. AOH resulted in an accumulation of cells in the G2/M-phase (4N). Most cells exhibited a large G2 nucleus whereas numbers of true mitotic cells were reduced relative to control. Both cyclin B1 and p-cdc2 levels increased, while cyclin B1 remained in the cytoplasm; suggesting arrest in the G2/M transition point. Remarkably, after exposure to AOH for 24h, most of the cells exhibited abnormally shaped nuclei, as evidenced by partly divided nuclei, nuclear blebs, polyploidy and micronuclei (MN). AOH treatment also induced abnormal Aurora B bridges, suggesting that cytokinesis was interfered within cells undergoing karyokinesis. A minor part of the resultant G1 tetraploid (4N) cells re-entered the S-phase and progressed to 8N cells.
ISSN: 0378-4274

inserm-00871659
http://www.hal.inserm.fr/inserm-00871659
http://www.hal.inserm.fr/inserm-00871659/document
http://www.hal.inserm.fr/inserm-00871659/file/Alternariol_induces_abnormal_nuclear_morphology_and_cell_cycle_arrest_in_murine_RAW_264.7_macrophages-accepted.pdf
DOI : 10.1016/j.toxlet.2013.02.012