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1.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360828

RESUMO

The environmental pollutant benzo[a]pyrene (BaP) is a human carcinogen that reacts with DNA after metabolic activation catalysed by cytochromes P450 (CYP) 1A1 and 1B1 together with microsomal epoxide hydrolase. The azo dye Sudan I is a potent inducer of CYP1A1/2. Here, Wistar rats were either treated with single doses of BaP (150 mg/kg bw) or Sudan I (50 mg/kg bw) alone or with both compounds in combination to explore BaP-derived DNA adduct formation in vivo. Using 32P-postlabelling, DNA adducts generated by BaP-7,8-dihydrodiol-9,10-epoxide were found in livers of rats treated with BaP alone or co-exposed to Sudan I. During co-exposure to Sudan I prior to BaP treatment, BaP-DNA adduct levels increased 2.1-fold in comparison to BaP treatment alone. Similarly, hepatic microsomes isolated from rats exposed to Sudan I prior to BaP treatment were also the most effective in generating DNA adducts in vitro with the activated metabolites BaP-7,8-dihydrodiol or BaP-9-ol as intermediates. DNA adduct formation correlated with changes in the expression and/or enzyme activities of CYP1A1, 1A2 and 1B1 in hepatic microsomes. Thus, BaP genotoxicity in rats in vivo appears to be related to the enhanced expression and/or activity of hepatic CYP1A1/2 and 1B1 caused by exposure of rats to the studied compounds. Our results indicate that the industrially employed azo dye Sudan I potentiates the genotoxicity of the human carcinogen BaP, and exposure to both substances at the same time seems to be hazardous to humans.


Assuntos
Benzo(a)pireno/toxicidade , Citocromo P-450 CYP1A1/metabolismo , Adutos de DNA/toxicidade , Fígado/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Naftóis/toxicidade , Animais , Carcinógenos Ambientais/toxicidade , Corantes/toxicidade , Masculino , Ratos , Ratos Wistar
2.
Arch Toxicol ; 95(6): 1917-1942, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34003343

RESUMO

Pyrrolizidine alkaloids (PAs) and PA N-oxides are common phytotoxins produced by over 6000 plant species. Humans are frequently exposed to PAs via ingestion of PA-containing herbal products or PA-contaminated foods. PAs require metabolic activation to form pyrrole-protein adducts and pyrrole-DNA adducts which lead to cytotoxicity and genotoxicity. Individual PAs differ in their metabolic activation patterns, which may cause significant difference in toxic potency of different PAs. This review discusses the current knowledge and recent advances of metabolic pathways of different PAs, especially the metabolic activation and metabolism-mediated cytotoxicity and genotoxicity, and the risk evaluation methods of PA exposure. In addition, this review provides perspectives of precision toxicity assessment strategies and biomarker development for the risk control and translational investigations of human intoxication by PAs.


Assuntos
Adutos de DNA/toxicidade , Dano ao DNA/efeitos dos fármacos , Alcaloides de Pirrolizidina/toxicidade , Animais , Biomarcadores/metabolismo , Adutos de DNA/química , Humanos , Mutagênicos/metabolismo , Mutagênicos/toxicidade , Alcaloides de Pirrolizidina/metabolismo , Medição de Risco/métodos
3.
Food Chem Toxicol ; 140: 111325, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32272200

RESUMO

As one of the most widespread environmental pollutants, benzo[α]pyrene is metabolized to diol epoxides and then covalently breaks the initial DNA base pairs, which has been closely related to the occurrence and development of many human cancers. High fidelity DNA polymerases play an extremely important role in maintaining the reliability or fidelity of nucleic acid replication, which is generally blocked by BP adducts. To reveal the blocking mechanism of BP, two comparative molecular dynamics simulations were performed for the thermophilic Bacillus stearothermophilus DNA polymerase I large fragment (BF) complexes with normal and BP-bound DNA duplexes. The results of global conformational changes and molecular interactions show that the association of BP leads to the rearrangement of intramolecular hydrogen bonds, impairing the molecular recognition between the polymerase and the DNA duplex. It is also found that the conformation of DNA duplex is distorted, accompanied by an increase in molecular overall rigidity. In terms of possible blocking mechanisms, the BP moiety perfectly integrates itself into the base-paired environment in a special vertical conformation and occupies the space required for the incoming nucleotide. This work provides useful dynamics and structural information for understanding the toxic effect of BP on DNA replication at atomic level.


Assuntos
Benzo(a)pireno/química , Adutos de DNA/química , DNA/efeitos dos fármacos , Desoxiguanosina/química , Benzo(a)pireno/toxicidade , Adutos de DNA/toxicidade , Humanos , Ligação de Hidrogênio , Reprodutibilidade dos Testes
4.
Mol Cell ; 74(2): 227-229, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31002804

RESUMO

In a recent issue of Science, Wilson et al. (2019) provide direct evidence that the bacterial-produced colibactin alkylates DNA in vivo, resulting in DNA adducts, which mediates its genotoxic effect. This work reinforces the role of colibactin-producing bacteria in colon cancer pathogenesis.


Assuntos
Neoplasias Colorretais/microbiologia , Escherichia coli/genética , Microbioma Gastrointestinal/genética , Peptídeos/toxicidade , Policetídeos/toxicidade , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Adutos de DNA/genética , Adutos de DNA/toxicidade , Dano ao DNA/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Mutagênicos/metabolismo , Mutagênicos/toxicidade , Mutação/efeitos dos fármacos , Mutação/genética , Peptídeos/genética
5.
Environ Mol Mutagen ; 60(1): 29-41, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30307653

RESUMO

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) were disposed directly into the Saguenay River of the St. Lawrence Estuary (SLE) by local aluminum smelters (Quebec, Canada) for 50 years (1926-1976). PAHs in the river sediments are likely etiologically related to gastrointestinal epithelial cancers observed in 7% of 156 mature (>19-year old) adult beluga found dead along the shorelines. Because DNA adduct formation provides a critical link between exposure and cancer induction, and because PAH-DNA adducts are chemically stable, we hypothesized that SLE beluga intestine would contain PAH-DNA adducts. Using an antiserum specific for DNA modified with several carcinogenic PAHs, we stained sections of paraffin-embedded intestine from 51 SLE beluga (0-63 years), 4 Cook Inlet (CI) Alaska beluga (0-26 years), and 20 beluga (0-46 years) living in Arctic areas (Eastern Beaufort Sea, Eastern Chukchi Sea, Point Lay Alaska) and aquaria, all with low PAH contamination. Stained sections showed nuclear light-to-dark pink color indicating the presence of PAH-DNA adducts concentrated in intestinal crypt epithelial lining cells. Scoring of whole tissue sections revealed higher values for the 51 SLE beluga, compared with the 20 Arctic and aquarium beluga (P = 0.003). The H-scoring system, applied to coded individual photomicrographs, confirmed that SLE beluga and CI beluga had levels of intestinal PAH-DNA adducts significantly higher than Arctic and aquarium beluga (P = 0.003 and 0.02, respectively). Furthermore, high levels of intestinal PAH-DNA adducts in four SLE beluga with gastrointestinal cancers, considered as a group, support a link of causality between PAH exposure and intestinal cancer in SLE beluga. Environ. Mol. Mutagen. 60:29-41, 2019. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.


Assuntos
Carcinogênese/induzido quimicamente , Adutos de DNA/toxicidade , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/patologia , Neoplasias Gastrointestinais/etiologia , Neoplasias Gastrointestinais/patologia , Mucosa Intestinal/patologia , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Animais , Regiões Árticas , Beluga , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Mucosa Intestinal/citologia , Camundongos , Poluentes Químicos da Água/toxicidade
6.
Invest New Drugs ; 37(2): 238-251, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29931584

RESUMO

Epidemiological surveys have revealed that environmental and dietary factors contribute to most of the human cancers. Our earlier studies have shown that resveratrol (RVT), a phytochemical reduced the tumor number, size and incidence of dysplasias induced by benzo(a)pyrene (BaP), an environmental toxicant in the ApcMin/+ mouse model of colon cancer. In this study we investigated to ascertain whether the preventive effects of RVT on BaP-induced colon carcinogenesis is a result of altered BaP biotransformation by RVT. For the first group of mice, 100 µg BaP/kg bw was administered in peanut oil via oral gavage over a 60 day period. For the second group, 45 µg RVT/kg bw was co-administered with BaP. For the third group, RVT was administered for 1 week prior to BaP exposure. Blood, colon and liver were collected from control and BaP/RVT-treated mice at 60 days post-BaP & RVT exposure. We have assayed activities and expression (protein & mRNA) of drug metabolizing enzymes such as cytochrome P4501A1 (CYP1A1), CYP1B1, and glutathione-S-transferase (GST) in colon and liver samples from the treatment groups mentioned above. An increased expression of CYP1A1 in liver and colon and of CYP1B1 in liver of BaP-treated mice was seen, while RVT inhibited the extent of biotransformation mediated by these enzymes in the respective tissue samples. In the case of GST, an increased expression in colon of BaP alone-treated mice was noted when RVT was administered prior to BaP or simultaneously with BaP. However, there is no change in liver GST expression between BaP and RVT treatment groups. The concentrations of BaP aqueous (phase II) metabolites were found to be greater than the organic (phase I) metabolites, suggesting that RVT slows down the phase I metabolism (metabolic activation) of BaP, while enhancing phase II metabolism (detoxification). Additionally, the BaP-DNA adduct concentrations measured in colon and liver of BaP + RVT-treated mice were low relative to their BaP counterparts. Taken together, our findings strongly suggest that RVT alleviates BaP-induced colon carcinogenesis by impairing biotransformation pathways and DNA adduct formation, and therefore holds promise as a chemopreventive agent.


Assuntos
Benzo(a)pireno/toxicidade , Biotransformação/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Resveratrol/farmacologia , Animais , Antioxidantes/farmacocinética , Antioxidantes/farmacologia , Apoptose , Benzo(a)pireno/química , Benzo(a)pireno/farmacocinética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinógenos Ambientais/química , Carcinógenos Ambientais/farmacocinética , Carcinógenos Ambientais/toxicidade , Proliferação de Células , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Adutos de DNA/química , Adutos de DNA/farmacocinética , Adutos de DNA/toxicidade , Glutationa Transferase/metabolismo , Humanos , Masculino , Camundongos , Resveratrol/farmacocinética , Distribuição Tecidual , Células Tumorais Cultivadas
7.
Chem Res Toxicol ; 31(9): 924-935, 2018 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-30169026

RESUMO

The most common lesion in DNA occurring due to clinical treatment with Temozolomide or cellular exposures to other methylating agents is 7-methylguanine (N7-Me-dG). It can undergo a secondary reaction to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-methylformamidopyrimidine (MeFapy-dG). MeFapy-dG undergoes epimerization in DNA to produce either α or ß deoxyribose anomers. Additionally, conformational rotation around the formyl bond, C5- N5 bond, and glycosidic bond may occur. To characterize and quantitate the mixture of these isomers in DNA, a 13C-MeFapy-dG lesion, in which the CH3 group of the MeFapy-dG was isotopically labeled, was incorporated into the trimer 5'-TXT-3' and the dodecamer 5'-CATXATGACGCT-3' (X = 13C-MeFapy-dG). NMR spectroscopy of both the trimer and dodecamer revealed that the MeFapy-dG lesion exists in single strand DNA as ten configurationally and conformationally discrete species, eight of which may be unequivocally assigned. In the duplex dodecamer, the MeFapy-dG lesion exists as six configurationally and conformationally discrete species. Analyses of NMR data in the single strand trimer confirm that for each deoxyribose anomer, atropisomerism occurs around the C5- N5 bond to produce R a and S a atropisomers. Each atropisomer exhibits geometrical isomerism about the formyl bond yielding E and Z conformations. 1H NMR experiments allow the relative abundances of the species to be determined. For the single strand trimer, the α and ß anomers exist in a 3:7 ratio, favoring the ß anomer. For the ß anomer, with respect to the C5- N5 bond, the R a and S a atropisomers are equally populated. However, the Z geometrical isomer of the formyl moiety is preferred. For the α anomer, the E- S a isomer is present at 12%, whereas all other isomers are present at 5-7%. DNA processing enzymes may differentially recognize different isomers of the MeFapy-dG lesion. Moreover, DNA sequence-specific differences in the populations of configurational and conformational species may modulate biological responses to the MeFapy-dG lesion.


Assuntos
Adutos de DNA/toxicidade , DNA/efeitos dos fármacos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Cromatografia Líquida de Alta Pressão/métodos , DNA/química , Dano ao DNA , Reparo do DNA , Replicação do DNA , Eletroforese Capilar/métodos , Isomerismo , Conformação de Ácido Nucleico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
8.
Environ Int ; 113: 335-340, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29395277

RESUMO

BACKGROUND: To examine the molecular benefits of the government action to close the local coal burning power plant in Tongliang County, Chongqing Municipality, we compared biologic markers and health outcomes in two successive birth cohorts enrolled before and after the plant was shut down. In this city, polycyclic aromatic hydrocarbons (PAH) were primarily emitted by the coal burning facility. We previously reported that cord blood levels of PAH-DNA adducts (a biomarker of exposure) and various adverse health outcomes were reduced in the second cohort, whereas levels of brain-derived neurotrophic factor/BDNF (a protein involved in neuronal growth) were increased. Here we assessed telomere length (TL), which has been associated with risk of certain chronic diseases, early mortality, aging and cognitive decline in adults. OBJECTIVES: The goals of the present study were to determine whether TL differed between the two cohorts and whether prenatal PAH exposure, estimated by PAH-DNA adducts in cord white blood cells of newborns in China, were predictive of shorter TL in cord blood, suggesting the potential accrual of risk of certain chronic diseases during the prenatal period. We explored relationships of TL with BDNF and neurodevelopmental outcomes, each previously associated with PAH-DNA adducts in these cohorts, as well as the potential mediating role of TL in the associations between adducts and neurodevelopmental outcomes. METHODS: We analyzed TL in cord blood of 255 newborns who also had data on PAH-DNA adducts, BDNF, and relevant covariates. Multiple regression analysis was carried out to test associations between adducts and TL and between TL and BDNF, adjusting for relevant covariates. In the subset with developmental quotient (DQ) scores from Gesell testing at age 2 (N = 210), we explored whether TL was a mediator of the relationship between PAH-DNA adducts and DQ scores by first examining the associations between cord adducts and DQ, cord adducts and TL, and TL and DQ, adjusting for the same covariates. RESULTS: As hypothesized, the mean TL was significantly higher in the second cohort compared to the first cohort. Overall, PAH-DNA cord adducts were significantly and inversely correlated with TL. Multiple regression analysis showed a significant association between adducts and TL, after adjusting for key covariates: ß (effect size per standard deviation adducts) = -0.019, p = .003. The regression coefficient of TL on (Ln) BDNF was also significant (ß = 0.167, p < .001). Exploratory analysis, regressing TL on Gesell developmental scores, showed generally inverse, but not significant associations. TL was not, therefore, deemed to be a potential mediator of the association between adducts and developmental scores at age two. CONCLUSION: This study provides the first evidence that prenatal PAH exposure from coal burning may adversely affect TL, with potential implications for future risk of chronic diseases including cardiovascular disease. The improvement in TL in the second cohort and the observed correlation between increased TL and higher levels of BDNF indicate direct benefits to the health and development of children resulting from the government's closure of the power plant.


Assuntos
Poluição do Ar/efeitos adversos , Adutos de DNA/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Efeitos Tardios da Exposição Pré-Natal/epidemiologia , Homeostase do Telômero/efeitos dos fármacos , Telômero/efeitos dos fármacos , Adulto , China/epidemiologia , Carvão Mineral , Estudos de Coortes , Adutos de DNA/análise , Feminino , Sangue Fetal , Humanos , Recém-Nascido , Masculino , Hidrocarbonetos Policíclicos Aromáticos/análise , Centrais Elétricas , Gravidez , Análise de Regressão
9.
Nat Rev Mol Cell Biol ; 18(9): 563-573, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28655905

RESUMO

Covalent DNA-protein crosslinks (DPCs, also known as protein adducts) of topoisomerases and other proteins with DNA are highly toxic DNA lesions. Of note, chemical agents that induce DPCs include widely used classes of chemotherapeutics. Their bulkiness blocks virtually every chromatin-based process and makes them intractable for repair by canonical repair pathways. Distinct DPC repair pathways employ unique points of attack and are crucial for the maintenance of genome stability. Tyrosyl-DNA phosphodiesterases (TDPs) directly hydrolyse the covalent linkage between protein and DNA. The MRE11-RAD50-NBS1 (MRN) nuclease complex targets the DNA component of DPCs, excising the fragment affected by the lesion, whereas proteases of the spartan (SPRTN)/weak suppressor of SMT3 protein 1 (Wss1) family target the protein component. Loss of these pathways renders cells sensitive to DPC-inducing chemotherapeutics, and DPC repair pathways are thus attractive targets for combination cancer therapy.


Assuntos
Adutos de DNA/toxicidade , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Animais , Antineoplásicos/efeitos adversos , Adutos de DNA/metabolismo , Instabilidade Genômica , Humanos , Neoplasias/tratamento farmacológico
10.
Artigo em Alemão | MEDLINE | ID: mdl-28516258

RESUMO

The assessment of health risks resulting from the intake of genotoxic carcinogens in food depends essentially on a valid exposure assessment. The reliability of the external exposure estimation is restricted by various factors, e. g. inaccurate data from dietary protocols and variations of food contaminant contents. As an alternative, the individual internal exposure to genotoxic substances may be described by specific biomarkers in different matrices. For example, mercapturic acids formed after glutathione conjugation of electrophilic metabolites can be detected in the urine. This typically reflects the exposure to the parent compound over a period of one to two days. The determination of adducts in the blood proteins serum albumin (SA) and hemoglobin (Hb) allows for conclusions to be drawn about the external exposure within the last three weeks (SA) or within the last four months (Hb). Protein adducts are used routinely in occupational medicine as biomarkers of internal exposure to substances in the ambient air of the workplace. The availability of increasingly sensitive analytical techniques also makes it possible to detect numerous adducts in proteins from human blood samples that are formed after the continuous intake of very small doses of toxic substances from foods. Here, we present the current state of science exemplified by protein adducts of the food contaminants acrylamide, aflatoxin B1 and glycidol. The biomarker can be used in the future to investigate previously unknown relationships between internal exposure and disease incidences.


Assuntos
Biomarcadores/análise , Contaminação de Alimentos/análise , Doenças Transmitidas por Alimentos/diagnóstico , Medição de Risco , Acetilcisteína/análise , Acetilcisteína/toxicidade , Carcinógenos/análise , Carcinógenos/toxicidade , Adutos de DNA/análise , Adutos de DNA/toxicidade , Alemanha , Análise de Perigos e Pontos Críticos de Controle , Humanos , Testes de Mutagenicidade , Mutagênicos/análise , Mutagênicos/toxicidade
11.
Carcinogenesis ; 38(7): 691-698, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28535209

RESUMO

Epidemiologic evidence linking environmental exposure to polycyclic aromatic hydrocarbons (PAH) with breast cancer is limited. Measurement of DNA adducts formed by aromatic compounds, including PAH, has been carried in breast tissue samples and white blood cells from women with breast cancer and different kinds of controls. However, these studies provide inconsistent results and bias cannot be ruled out. During the 7-year follow-up period, 305 women were diagnosed with first primary breast cancer in the EPIC-Spain cohort, and were compared with a sample of 149 women without breast cancer at recruitment, using a case-cohort approach. Aromatic adducts to DNA from leukocytes collected at recruitment were measured by means of the 32P-post-labelling technique. The relative risk and 95% confidence interval (CI), adjusted by relevant confounders, were estimated by a modified version of Cox proportional hazards model. There was a significant increased risk for developing breast cancer when DNA adduct concentrations were doubled, with adjusted RR of 1.61 (95% CI 1.29-2.01). The increase in breast cancer risk was observed both for pre- and post-menopausal women. There was a significant interaction with tobacco smoking and body mass index, with higher effect of DNA adducts on breast cancer risk among smokers and women with normal weight. The results from our study support the hypothesis that factors leading to higher levels of aromatic DNA adducts in white blood cells may be involved in development of breast cancer.


Assuntos
Neoplasias da Mama/genética , Adutos de DNA/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Adulto , Idoso , Índice de Massa Corporal , Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/patologia , Estudos de Coortes , Adutos de DNA/genética , Exposição Ambiental , Feminino , Humanos , Leucócitos , Pessoa de Meia-Idade , Fatores de Risco , Fumar/efeitos adversos , Espanha
12.
Biochem J ; 474(11): 1837-1852, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28408432

RESUMO

1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein - an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A → C and A → T transversions and, less frequently, A → G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II), and αKG concentrations for this reaction were determined. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S stereoisomer seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA.


Assuntos
Adenina/análogos & derivados , Enzimas AlkB/metabolismo , Carcinógenos Ambientais/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutagênicos/metabolismo , Adenina/química , Adenina/metabolismo , Adenina/toxicidade , Enzimas AlkB/química , Enzimas AlkB/genética , Sítios de Ligação , Biocatálise , Carcinógenos Ambientais/química , Carcinógenos Ambientais/toxicidade , Adutos de DNA/química , Adutos de DNA/toxicidade , DNA Bacteriano/química , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Estabilidade Enzimática , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidroxilação , Conformação Molecular , Simulação de Dinâmica Molecular , Mutagênese/efeitos dos fármacos , Mutagênicos/química , Mutagênicos/toxicidade , Oxirredução , Conformação Proteica , Teoria Quântica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato
13.
Proc Natl Acad Sci U S A ; 114(15): E3101-E3109, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28351974

RESUMO

Aflatoxin B1 (AFB1) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB1-DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB1 using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB1 exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB1 exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB1, and, as such, is an early detection metric for AFB1-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.


Assuntos
Aflatoxina B1/genética , Biomarcadores/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Adutos de DNA/genética , Neoplasias Hepáticas/genética , Mutação , Aflatoxina B1/toxicidade , Animais , Carcinogênese/induzido quimicamente , Carcinogênese/patologia , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Adutos de DNA/toxicidade , Feminino , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL
14.
Basic Clin Pharmacol Toxicol ; 121 Suppl 3: 63-77, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28032943

RESUMO

DNA-protein cross-links (DPCs) are unusually bulky DNA adducts that form in cells as a result of exposure to endogenous and exogenous agents including reactive oxygen species, ultraviolet light, ionizing radiation, environmental agents (e.g. transition metals, formaldehyde, 1,2-dibromoethane, 1,3-butadiene) and common chemotherapeutic agents. Covalent DPCs are cytotoxic and mutagenic due to their ability to interfere with faithful DNA replication and to prevent accurate gene expression. Key to our understanding of the biological significance of DPC formation is identifying the proteins most susceptible to forming these unusually bulky and complex lesions and quantifying the extent of DNA-protein cross-linking in cells and tissues. Recent advances in bottom-up mass spectrometry-based proteomics have allowed for an unbiased assessment of the whole protein DPC adductome after in vitro and in vivo exposures to cross-linking agents. This MiniReview summarizes current and emerging methods for DPC isolation and analysis by mass spectrometry-based proteomics. We also highlight several examples of successful applications of these novel methodologies to studies of DPC lesions induced by bis-electrophiles such as formaldehyde, 1,2,3,4-diepoxybutane, nitrogen mustards and cisplatin.


Assuntos
Reagentes de Ligações Cruzadas/toxicidade , Adutos de DNA/análise , Exposição Ambiental/efeitos adversos , Espectrometria de Massas/métodos , Cisplatino/toxicidade , DNA/química , Adutos de DNA/química , Adutos de DNA/toxicidade , Dano ao DNA , Compostos de Epóxi/toxicidade , Formaldeído/toxicidade , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Compostos de Mostarda Nitrogenada/toxicidade , Proteínas/química , Proteômica/métodos
15.
Basic Clin Pharmacol Toxicol ; 121 Suppl 3: 44-54, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27889941

RESUMO

Human beings are exposed to many reactive electrophiles, both formed endogenously and from exogenous exposures. Such compounds could react with cellular biomolecules and form stable reaction products, adducts, at nucleophilic sites in proteins and DNA, constituting a risk for toxic effects. Adductomic approaches aim to study the totality of adducts, to specific biomolecules, by mass spectrometric screening. This Mini-Review focuses on the development and application of an adductomic approach for the screening of unknown adducts to N-terminal valine (Val) in haemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC-MS/MS). The approach is based on the FIRE procedure, a modified Edman procedure for the analysis of adducts to N-terminal Val in Hb by LC-MS/MS. In the first application of the approach, samples from 12 smokers/non-smokers were screened for Hb adducts, and six previously identified adducts and 20 unknown adducts were detected. To confirm the observation of the detected unknown adducts, targeted screenings were performed in larger sets of blood samples (n = 50-120) from human cohorts. The majority of the previously detected unknown adducts was found in all analysed samples, with large interindividual variations in adduct levels. For structural identification of unknown adducts, a strategy using adductome LC-MS/MS data was formulated and applied. Six identified adducts correspond to ethylation and the precursor electrophiles ethyl vinyl ketone, glyoxal, methylglyoxal, acrylic acid and 1-octen-3-one. The observation of these adducts in human blood motivate further studies to evaluate possible contributions to health risks, as well as their potential as biomarkers of exposure.


Assuntos
Adutos de DNA/análise , Exposição Ambiental/efeitos adversos , Hemoglobinas/análise , Valina/análise , Aldeídos/análise , Aldeídos/química , Aldeídos/toxicidade , Biomarcadores/análise , Cromatografia Líquida/métodos , Adutos de DNA/química , Adutos de DNA/toxicidade , Compostos de Epóxi/análise , Compostos de Epóxi/química , Compostos de Epóxi/toxicidade , Hemoglobinas/química , Humanos , Nitrosaminas/análise , Nitrosaminas/química , Nitrosaminas/toxicidade , Espectrometria de Massas em Tandem/métodos , Valina/química
16.
Chem Res Toxicol ; 29(9): 1493-503, 2016 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-27404553

RESUMO

The generation of chemical alkylating agents from nitrosation of glycine and bile acid conjugates in the gastrointestinal tract is hypothesized to initiate carcinogenesis. O(6)-carboxymethylguanine (O(6)-CMG) is a product of DNA alkylation derived from nitrosated glycine. Although the tendency of the structurally related adduct O(6)-methylguanine to code for the misincoporation of TTP during DNA replication is well-established, the impact of the presence of the O(6)-CMG adduct in a DNA template on the efficiency and fidelity of translesion DNA synthesis (TLS) by human DNA polymerases (Pols) has hitherto not been described. Herein, we characterize the ability of the four human TLS Pols η, ι, κ, and ζ and the replicative Pol δ to bypass O(6)-CMG in a prevalent mutational hot-spot for colon cancer. The results indicate that Pol η replicates past O(6)-CMG, incorporating dCMP or dAMP, whereas Pol κ incorporates dCMP only, and Pol ι incorporates primarily dTMP. Additionally, the subsequent extension step was carried out with high efficiency by TLS Pols η, κ, and ζ, while Pol ι was unable to extend from a terminal mismatch. These results provide a first basis of O(6)-CMG-promoted base misincorporation by Y- and B-family polymerases potentially leading to mutational signatures associated with colon cancer.


Assuntos
Adutos de DNA/química , Adutos de DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/análogos & derivados , Guanina/química , Adutos de DNA/toxicidade , DNA Polimerase Dirigida por DNA/química , Guanina/toxicidade , Humanos , Mutagênicos/química , Mutagênicos/metabolismo , Mutagênicos/toxicidade , Mutação , Nitrosação , Proteínas Proto-Oncogênicas p21(ras)/genética
17.
Toxicol Appl Pharmacol ; 290: 31-42, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26603514

RESUMO

Test batteries to screen chemicals for mutagenic hazard include several endpoints regarded as effective for detecting genotoxic carcinogens. Traditional in vivo methods primarily examine clastogenic endpoints in haematopoietic tissues. Although this approach is effective for identifying systemically distributed clastogens, some mutagens may not induce clastogenic effects; moreover, genotoxic effects may be restricted to the site of contact and/or related tissues. An OECD test guideline for transgenic rodent (TGR) gene mutation assays was released in 2011, and the TGR assays permit assessment of mutagenicity in any tissue. This study assessed the responses of two genotoxicity endpoints following sub-chronic oral exposures of male Muta™Mouse to 9 carcinogenic polycyclic aromatic hydrocarbons (PAHs). Clastogenicity was assessed via induction of micronuclei in peripheral blood, and mutagenicity via induction of lacZ transgene mutations in bone marrow, glandular stomach, small intestine, liver, and lung. Additionally, the presence of bulky PAH-DNA adducts was examined. Five of the 9 PAHs elicited positive results across all endpoints in at least one tissue, and no PAHs were negative or equivocal across all endpoints. All PAHs were positive for lacZ mutations in at least one tissue (sensitivity=100%), and for 8 PAHs, one or more initial sites of chemical contact (i.e., glandular stomach, liver, small intestine) yielded a greater response than bone marrow. Five PAHs were positive in the micronucleus assay (sensitivity=56%). Furthermore, all PAHs produced DNA adducts in at least one tissue. The results demonstrate the utility of the TGR assay for mutagenicity assessment, especially for compounds that may not be systemically distributed.


Assuntos
Adutos de DNA/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Testes de Toxicidade/métodos , Animais , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Óperon Lac/genética , Masculino , Camundongos , Camundongos Transgênicos , Testes para Micronúcleos , Mutagênicos/toxicidade , Mutação , Sensibilidade e Especificidade
18.
Arch Toxicol ; 90(10): 2461-80, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26377693

RESUMO

In this study, the accuracy of the assumption that genotoxic, carcinogenic polycyclic aromatic hydrocarbons (PAHs) act via similar mechanisms of action as benzo(a)pyrene (BaP), the reference PAH used in the human health risk assessment of PAH-containing complex mixtures, was investigated. Adult male Muta™Mouse were gavaged for 28 days with seven individual, genotoxic PAHs. Global gene expression profiles in forestomach, liver, and lung (target tissues of exposure) were determined at 3 days post-exposure. The results are compared with our previously published results from mice exposed to BaP via the same exposure regimen. Although all PAHs showed enhanced ethoxyresorufin-O-deethylase activity, DNA adduct formation, and lacZ mutant frequency in the lungs, the unsupervised cluster analysis of differentially expressed genes revealed that the transcriptional changes are both PAH- and tissue-specific, with lung showing the most response. Further bioinformatics-/pathway-based analysis revealed that all PAHs induce expression of genes associated with carcinogenic processes, including DNA damage response, immune/inflammatory response, or cell signaling processes; however, the type of pathways and the magnitude of change varied for each PAH and were not the same as those observed for BaP. Benchmark dose modeling showed transcriptomic data closely reflected the known tumor incidence for the individual PAHs in each tissue. Collectively, the results suggest that the underlying mechanisms of PAH-induced toxicity leading to tumorigenesis are tissue-specific and not the same for all PAHs; based on the tissue type considered, use of BaP as a reference chemical may overestimate or underestimate the carcinogenic potential of PAHs.


Assuntos
Carcinógenos Ambientais/toxicidade , Adutos de DNA/toxicidade , Mutagênicos/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Transcriptoma/efeitos dos fármacos , Animais , Benzo(a)pireno/toxicidade , Análise por Conglomerados , Mucosa Gástrica/metabolismo , Óperon Lac/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Transgênicos , Estômago/efeitos dos fármacos , Estômago/patologia , Toxicogenética
19.
Biomolecules ; 5(4): 2987-3008, 2015 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-26556381

RESUMO

Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease.


Assuntos
Aldeídos/toxicidade , Adutos de DNA/toxicidade , Etanol/toxicidade , Doenças Respiratórias/induzido quimicamente , Fumar/efeitos adversos , Poluição por Fumaça de Tabaco/efeitos adversos , Aldeídos/metabolismo , Animais , Adutos de DNA/metabolismo , Humanos , Doenças Respiratórias/metabolismo
20.
Chem Res Toxicol ; 28(12): 2250-2, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26544157

RESUMO

This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 10(8) nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 10(8) nucleotides per hour in carboplatin alone (p = 0.021). This rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.


Assuntos
Carboplatina/metabolismo , Adutos de DNA/metabolismo , Paclitaxel/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Carboplatina/uso terapêutico , Carboplatina/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Adutos de DNA/uso terapêutico , Adutos de DNA/toxicidade , Sinergismo Farmacológico , Humanos , Paclitaxel/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico
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