RESUMO
1-Methylpyrene (1-MP) is a common environmental pollutant and animal carcinogen. After sequential activation by cytochromes P450 and sulfotransferases, it induced gene mutations and micronuclei in mammalian cells. The type of micronuclei formed, entire chromosomes or fragments, was not analysed. In this study, 1-MP and its primary metabolite, 1-hydroxymethylpyrene (1-HMP), were investigated for the induction of centromere-positive and -negative micronuclei in the human hepatoma cell line HepG2 and its derivative C3A, expressing relevant enzymes at higher levels. Under a short-exposure (9 h)/long-recovery regime (2 cell cycles in total), 1-MP and 1-HMP provided negative test results in HepG2 cells. However, they induced micronuclei in C3A cells, the effect being blocked by 1-aminobenzotriazole (inhibitor of cytochromes P450s) and reduced by pentachlorophenol (inhibitor of sulfotransferases). Immunofluorescence staining of centromere protein B in the micronuclei revealed purely clastogenic effects under this regime. Unexpectedly, 1-MP and 1-HMP at concentrations 1/5-1/4 of that required for micronuclei formation led to mitotic arrest and spindle aberrations, as detected by immunofluorescence staining of ß- and γ-tubulin. Following extended exposure (72 h, 2 cell cycles, no recovery), damage to the spindle apparatus and centrosomes was detected at even lower concentrations, with concurrent formation of micronuclei. At low concentrations (1-8 µM 1-MP, 0.25-0.5 µM 1-HMP), the micronuclei induced were unexceptionally centromere-positive. Thus, the chromosome-damaging mechanism of 1-MP was regime and concentration dependent: potently aneugenic under persistent exposure, while clastogenic at higher concentrations following a short-exposure/long-recovery regime. This is a convincing evidence for the existence of metabolic activation-dependent aneugens.
Assuntos
Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Mitose/efeitos dos fármacos , Pirenos/toxicidade , Ativação Metabólica/efeitos dos fármacos , Aneugênicos/metabolismo , Aneugênicos/toxicidade , Linhagem Celular Tumoral , Proteína B de Centrômero/metabolismo , Centrossomo/efeitos dos fármacos , Células Hep G2 , Humanos , Testes para Micronúcleos , Microscopia de Fluorescência , Mutagênicos , Pirenos/metabolismo , Fuso Acromático/efeitos dos fármacosRESUMO
Bisphenols (BPs) are environmental pollutants with relevant DNA damage in human population; however, they are generally inactive in standard mutagenicity assays, possibly due to insufficient metabolic activation. In this study, induction of micronuclei and double-strand DNA breaks by BPA, BPF, and BPS in Chinese hamster V79-derived cell lines expressing various human CYP enzymes and a human hepatoma (C3A) (metabolism-proficient) cell line were investigated. Molecular docking of BPs to human CYPs indicated some substrate-enzyme potentials, including CYP1A1 for each compound, which did not induce micronuclei in V79-derived cell lines expressing human CYP1A2, 2E1, or 3A4 but became positive in human CYP1A1-expressing (V79-hCYP1A1) cells. In V79-hCYP1A1 and C3A cells, all compounds induced double-strand DNA breaks and micronuclei formation, which were blocked/significantly attenuated by 1-aminobenzotriazole (CYP inhibitor) or 7-hydroxyflavone (selective CYP1A1 inhibitor). Coexposure of C3A cells to pentachlorophenol (sulfotransferase 1 inhibitor) or ketoconazole (UDP-glucuronosyltransferase 1A inhibitor) potentiated micronuclei induction by each compound, with thresholds lowered from 2.5-5.0 to 0.6-1.2 µM. Immunofluorescence staining of centromere protein B with micronuclei formed in C3A cells by each compound indicated pure clastogenic effects. In conclusion, BPs are potently clastogenic in mammalian cells, which require activation primarily by human CYP1A1 and are negatively modulated by phase II metabolism.
Assuntos
Citocromo P-450 CYP1A1 , Mutagênicos , Animais , Linhagem Celular , Cricetinae , Cricetulus , Citocromo P-450 CYP1A1/genética , Dano ao DNA , Humanos , Simulação de Acoplamento MolecularRESUMO
The PWWP domain of hepatoma-derived growth factor-related protein 2 (HDGFRP2) recognizes methylated histones to initiate the recruitment of homologous recombination repair proteins to damaged silent genes. The combined depletion of HDGFRP2 and its paralog PSIP1 effectively impedes the onset and progression of diffuse intrinsic pontine glioma (DIPG). Here, we discovered varenicline and 4-(4-bromo-1H-pyrazol-3-yl) pyridine (BPP) as inhibitors of the HDGFRP2 PWWP domain through a fragment-based screening method. The complex crystal structures reveal that both Varenicline and BPP engage with the aromatic cage of the HDGFRP2 PWWP domain, albeit via unique binding mechanisms. Notably, BPP represents the first single-digit micromolar inhibitor of the HDGFRP2 PWWP domain with a high ligand efficiency. As a dual inhibitor targeting both HDGFRP2 and PSIP1 PWWP domains, BPP offers an exceptional foundation for further optimization into a chemical tool to dissect the synergetic function of HDGFRP2 and PSIP1 in DIPG pathogenesis.
RESUMO
The composition of amino acids forming the active site of a CYP enzyme is impactful in its substrate selectivity. For CYP2E1, the role of PHE residues in the formation of effective binding orientations for its aromatic substrates remains unclear. In this study, molecular docking and molecular dynamics analysis were performed to reflect the interactions between PHEs in the active site of human CYP2E1 and various aromatic compounds known as its substrates. The results indicated that the orientation of 1-methylpyrene (1-MP) in the active site was highly determined by the presence of PHEs, PHE478 contributing to the binding free energy most significantly. Moreover, by building a random forest model the relationship between each of 19 molecular descriptors of polychlorinated biphenyl (PCB) compounds (from molecular docking, quantum mechanics, and physicochemical properties) and their human CYP2E1-dependent mutagenicityas established mostly in our lab, was investigated. The presence of PHEs did not appear to significantly modify the electronic or structural feature of each bound ligand (PCB), instead, the flexibility of the conformation of PHEs contributed substantially to the effective binding energy and orientation. It is supposed that PHE residues adjust their own conformation to permit a suitablly shaped cavity for holding the ligand and forming its orientation as favorable for a biochemical reaction. This study has provided some insights into the role of PHEs in guiding the interactive adaptation of the active site of human CYP2E1 for the binding and metabolism of aromatic substrates.
RESUMO
The accessibility of reactive metabolites to test cells is critical for a genotoxic response. However, sulfo-conjugates formed outside may not readily enter cells, and some metabolites formed by cytochromes P450 (CYPs) may not endure transport. This topic was addressed in the present study, using V79 cells engineered for human CYPs and/or a sulfotransferase (SULT). First, 1-methylpyrene, 1-hydroxymethylpyrene, benzo[a]pyrene, and aflatoxin B1 significantly induced micronuclei in V79-hCYP1A2-hSULT1A1, V79-hSULT1A1, V79-hCYP1A1, and V79-hCYP1A2 cells, respectively. Subsequently, we used these cell lines as external activating systems in various experimental settings in combination with V79-derived target cells lacking critical enzymes. 1-Methylpyrene (activated by CYPs and SULTs sequentially) showed an activity similar to that in V79-hCYP1A2-hSULT1A1 cells, in each following model: a mixed V79-hCYP1A2:V79-hSULT1A1 (1:1) culture, exposure of V79-hCYP1A2 to 1-methylpyrene followed by transfer of medium to V79-hSULT1A1 target cells, and V79-hSULT1A1 communicating with V79-hCYP1A2 through 0.4-µm pores and over a 1-mm distance in a unique transwell system. These results suggest ready transfer of 1-hydroxymethylpyrene formed in V79-hCYP1A2 to V79-hSULT1A1 for further activation. In the last two models, with V79-hSULT1A1 for activation and V79-Mz as target, 1-hydroxymethylpyrene induced micronuclei mildly, suggesting limited intercellular transfer of the ultimate genotoxicant, 1-sulfooxymethylpyrene. Benzo[a]pyrene induced micronuclei in V79-Mz communicating with V79-hCYP1A1 via porous membranes, whereas aflatoxin B1 was inactive in V79-Mz communicating with V79-hCYP1A2. Our results suggest that the sulfo-conjugate tested may have difficulty entering cells for a genotoxic effect, and the reactive metabolite of aflatoxin B1, unlike that of benzo[a]pyrene, could not travel an adequate distance to enter cells. Environ. Mol. Mutagen. 61:224-234, 2020. © 2019 Wiley Periodicals, Inc.
Assuntos
Aflatoxina B1/toxicidade , Benzo(a)pireno/toxicidade , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mutagênicos/toxicidade , Pirenos/toxicidade , Aflatoxina B1/metabolismo , Animais , Arilsulfotransferase/metabolismo , Benzo(a)pireno/metabolismo , Linhagem Celular , Cricetinae , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Testes para Micronúcleos , Mutagênicos/metabolismo , Pirenos/metabolismoRESUMO
1-Methylpyrene (1-MP) is a ubiquitous environmental pollutant and rodent carcinogen. Its mutagenic activity depends on sequential activation by various CYP and sulfotransferase (SULT) enzymes. Previously we have observed induction of micronuclei and mitotic arrest by 1-MP in a Chinese hamster (V79)-derived cell line expressing both human CYP1A2 and SULT1A1 (V79-hCYP1A2-hSULT1A1), however, the mode of chromosome damage and the involvement of mitotic tubulin structures have not been clarified. In this study, we used immunofluorescent staining of centromere protein B (CENP-B) with the formed micronuclei, and that of ß- and γ-tubulin reflecting the structures of mitotic spindle and centrioles, respectively, in V79-hCYP1A2-hSULT1A1 cells. The results indicated that 1-MP induced micronuclei in V79-hCYP1A2-hSULT1A1 cells from 0.125 to 2 µM under a 24 h/0 h (exposure/recovery) regime, while in the parental V79-Mz cells micronuclei were induced by 1-MP only at concentrations ≥ 8 µM; in both cases, the micronuclei induced by 1-MP were predominantly CENP-B positive. Following 54 h of exposure, 1-MP induced mitotic spindle non-congression and centrosome amplification (multipolar mitosis) in V79-hCYP1A2-hSULT1A1 cells, and anaphase/telophase retardation, at concentrations ≥ 0.125 µM with concentration-dependence; while in V79-Mz cells it was inactive up to 8 µM. This study suggests that in mammalian cells proficient in activating enzymes 1-MP may induce chromosome loss and mitotic disturbance, probably by interfering with the mitotic spindle and centrioles.
Assuntos
Arilsulfotransferase/metabolismo , Cromossomos de Mamíferos/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Mitose/efeitos dos fármacos , Pirenos/farmacologia , Animais , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteína B de Centrômero/metabolismo , Cricetinae , Humanos , Micronúcleo Germinativo/efeitos dos fármacos , Micronúcleo Germinativo/metabolismo , Índice Mitótico , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismoRESUMO
Benzene is a human carcinogen that requires metabolic activation. We previously observed that benzene and its hydroxylated metabolites induce micronuclei in mammalian cells expressing human CYP2E1. This study was initially aimed to study another endpoint, the induction of gene mutations by those compounds in the same cell models. A V79-derived cell line expressing human CYP2E1 and sulfotransferase (SULT) 1A1 (V79-hCYP2E1-hSULT1A1) pretreated with ethanol (a CYP2E1 stabilizer) was used in the hprt gene mutagenicity assay. Phenol, hydroquinone, catechol, and 1,2,4-trihydroxybenzene all induced gene mutations, while they were inactive, or only weakly positive (hydroquinone), in parental V79-Mz cells. Unexpectedly, benzene was non-mutagenic in both cell lines, but it became positive in V79-hCYP2E1-hSULT1A1 cells using regimes of short exposure/long recovery without ethanol pretreatment, for both gene mutations and micronuclei formation. In silico molecular simulation showed binding energies and positions favorable for each compound to be oxidized by human CYP2E1, benzene demonstrating the highest affinity. By tunnel analysis, ethanol binding did not limit benzene to pass tunnel S, which was specifically active for benzene. However, its end product, acetic acid, decreased the occurrence of tunnel S from 5.4 to 2.2% and extended the length of its bottleneck from 5.5 to 9.0 Å. With residual ethanol molecules still being present in CYP2E1 for a period of time after benzene exposure, the acetic acid formed could limit the entrance of benzene, thus inhibit its metabolic activation. In summary, ethanol may interfere with the activation of benzene to mutagenic metabolites, at least in cultured cells.
Assuntos
Benzeno/toxicidade , Citocromo P-450 CYP2E1/metabolismo , Etanol/metabolismo , Mutagênicos/toxicidade , Arilsulfotransferase/metabolismo , Benzeno/metabolismo , Linhagem Celular , Citocromo P-450 CYP2E1/química , Humanos , Hidroxilação , Testes para Micronúcleos , Mutagênese/efeitos dos fármacos , Testes de Mutagenicidade , Mutagênicos/metabolismoRESUMO
Polychlorinated biphenyls (PCBs) are persistent organic pollutants with human carcinogenicity. Many lower chlorinated and non-dioxin-like PCBs have been observed to be mutagenic following activation by human CYP2E1, while activation of dioxin-like (DL-) PCBs by this enzyme has never been evidenced. In this study, each DL-PCB was analyzed by molecular docking to human CYP2E1 protein for predicting a substrate interaction. All compounds demonstrated high affinities with the active site of human CYP2E1, binding energy being -8.7 â¼ -9.7 kcal/mol. However, most compounds demonstrated ligand-heme distances as ≥ 6.8 Å, while the values for 2,3,3',4,4'- (PCB 105) and 2,3',4,4',5-pentachlorobiphenyl (PCB 118) were 5.3 and 5.4 Å, respectively (valid for electron transfer). Experimentally, both PCB 105 and 118 induced micronuclei in a V79-derived cell line engineered for expression of human CYP2E1 at low micromolar concentrations, while inactive or weakly positive in V79-Mz control cells; these effects were blocked or reduced by 1-aminobenzotriazole, a suicide CYP inhibitor. However, DL-PCBs 77, 81 and 126 were all negative in both cell lines. In a human hepatoma (C3A) cell line, PCB 105 and 118 induced micronuclei marginally, while with ethanol pretreatment (to stabilize CYP2E1) both compounds induced micronuclei efficiently, and co-exposure to trans-1,2-dichloroethylene (a selective CYP2E1 inhibitor) led to clearly negative results with both compounds. Finally, both PCB 105 and 118 induced PIG-A gene mutations in C3A cells, which was blocked by trans-1,2-dichloroethylene. In summary, in silico and experimental results consistently suggest that DL- PCBs 105 and 118 may be activated by human CYP2E1 for mutagenic activities.
Assuntos
Citocromo P-450 CYP2E1/metabolismo , Proteínas de Membrana/genética , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Simulação de Acoplamento Molecular , Mutação , Bifenilos Policlorados/toxicidade , Ativação Metabólica , Animais , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Domínio Catalítico , Cricetulus , Citocromo P-450 CYP2E1/química , Citocromo P-450 CYP2E1/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/patologia , Bifenilos Policlorados/química , Bifenilos Policlorados/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
We analyzed the expression of miR-503 in osteosarcoma tissues (OS) and discussed the clinical significance of our findings. To provide a theoretical basis for clinical applications, prognosis prediction and treatment of osteosarcoma, we studied the biological function of miR-503 and its mechanism in MG63 osteosarcoma cells. Real-time polymerase chain reaction (PCR) was used to detect the expression of miR-503 in 45 OS tissues and 20 osteochondroma tumors, analyzing the relationship between clinical pathology and follow-up data. Cox multivariate analysis revealed the clinical and pathological features of the osteosarcoma index and the influence of miR-503 expression on OS prognosis. To observe the effect on cell proliferation and invasion, MG-63 cells were transfected with miR-503. The TargetScan and PicTar bioinformatics method was used to analyze the probable target gene of miR-503 and, combined with the function of the target genes, resulted in a final validation of related pathways. miR-503 was significantly down-regulated in primary OS samples (26/45, 57.8%). The median miR-503 expression level in osteosarcoma was two-fold lower than that in osteochondroma (median expression 6.4 and 13.09, respectively, P< 0.05). The less-expressed miR-503 was associated with Enneking stage (p= 0.004) and invasion (p= 0.015) of OC. Patients with low miR-503 expression had poorer overall survival time. In the multivariate analysis, miR-503 was a significant prognostic factor (P= 0.010). miR-503 can inhibit proliferation and invasion in the MG63 cell line. Using bioinformatics, VEGFA and Rictor were determined to be the likely downstream target genes of miR-503. VEGFA, Rictor, Akt and Erk1/2 were negatively regulated by the overexpression of miR-503. In conclusion, miR-503 has significant tumor-suppressor biological activity and is thus likely to become a new target for the treatment of osteosarcoma.
Assuntos
MicroRNAs/genética , Osteossarcoma/genética , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Fator A de Crescimento do Endotélio Vascular/genética , Adolescente , Adulto , Apoptose/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteína Oncogênica v-akt/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Prognóstico , Adulto JovemRESUMO
The toxicity and persistence of perfluoroalkyl acids (PFAAs) in humans have drawn growing concerns, particularly for children. However, data regarding the concentrations of PFAAs in children are limited. In this study, we measured the concentrations of 14 PFAAs in plasma samples collected from 1192 children aged 0-7years from 7 cities in Guangdong Province: Guangzhou, Shenzhen, Foshan, Dongguan, Zhaoqing, Zhongshan and Zhanjiang. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were detected in >99.5% of the analysed samples. PFOS had the highest median concentration (23.6ng/mL) in the total samples, followed by PFOA (2.8ng/mL). The median concentrations of the other PFAAs were lower than 0.4ng/mL. The concentrations of perfluorohexanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluorododecanoic acid, perfluorohexane sulfonate, PFOA and PFOS in children from Foshan were significantly higher than those found in other cities (p<0.001). Negative correlations between most of the PFAA concentrations and age (r=-0.06--0.45) were found in all children. Weak to moderate correlations (r=0.080-0.698) were observed between all PFAA concentrations. Our findings indicated a high exposure of children to PFAAs in the early life-stage. The exposure sources and pathways of PFAAs in different regions are different. Considering a lack of information on the exposure pathways and health status, more studies are needed to evaluate the exposure resources and assess the health risk of PFAA exposure in children.
RESUMO
To evaluate the exposure of children to 14 perfluorinated compounds (PFCs) in a typical and representative industrial city, plasma samples from 476 children aged 0-7 years in Foshan, China, were analysed. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were detected in 100% of the samples, accounting for 82.27 and 11.46% of the total PFC concentrations, respectively, while PFOS peaked at age 0-1 years, for which the mean and median concentrations were 113.71 and 83.65 ng/mL, respectively, while PFOA peaked at age 3-4 years, for which the mean and median concentrations were 10.68 and 6.58 ng/mL, respectively. The concentrations of PFOS, perfluorohexane sulfonate and perfluorohexanoic acid decreased with age among children aged 0-7 years, and no gender-related differences were found in the concentrations of PFCs. A high correlation was found among all PFCs, especially between PFCs of similar carbon chains (r = 0.161-0.695, p < 0.05). In addition, the concentrations of PFOS and PFOA in children's plasma in Foshan were up to 40-fold higher than those reported in China and other countries. In conclusion, children in Foshan have extensive exposure to PFCs, especially in infancy. Further studies are needed to explore the impact of PFCs on children who live in a typical and representative industrial city in China. Graphical abstract á .