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1.
J Mater Chem B ; 8(43): 9951-9960, 2020 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-33034309

RESUMEN

A 2D CuNi metal-organic framework (MOF) named CuxNi3-x(HHTP)2 was synthesized with 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) as the linker and was used as a sensitive scaffold to adsorb aptamer strands for the electrochemical detection of living C6 glioma cells and one of their biomarkers, epidermal growth factor receptor (EGFR). Different from conventional MOFs, the CuxNi3-x(HHTP)2 MOF comprises long-range delocalized electrons, a graphene-analog nanostructure, multiple metal states (Cu0/Cu+/Cu2+ and Ni2+/Ni3+), and abundant oxygen vacancies. With these features, the CuxNi3-x(HHTP)2 MOF anchored a large amount of C6 cell-targeted aptamer strands via coordination among metal centers, oligonucleotides, π-π stacking, and van der Waals force. The CuxNi3-x(HHTP)2-based cytosensor showed a low limit of detection (LOD) of 21 cells mL-1 toward C6 glioma cells within a wide range from 50 cells mL-1 to 1 × 105 cells mL-1. Moreover, the proposed aptasensor displayed high selectivity, good stability, acceptable reproducibility, and a low LOD of 0.72 fg mL-1 for detecting EGFR with the concentration ranging from 1 fg mL-1 to 1 ng mL-1. The aptasensor based on the CuxNi3-x(HHTP)2 MOF exhibited superior sensing performance over those based on its monometallic analogues such as Cu3(HHTP)2 MOF and Ni3(HHTP)2 MOF. Hence, this sensing strategy based on a bimetallic semiconducting MOF shows great potential for cancer diagnosis.


Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Neoplasias Encefálicas/diagnóstico , Glioma/diagnóstico , Estructuras Metalorgánicas/química , Línea Celular Tumoral , Crisenos/química , Cobre/química , Técnicas Electroquímicas/métodos , Receptores ErbB/análisis , Humanos , Níquel/química , Semiconductores
2.
DNA Repair (Amst) ; 95: 102935, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32721818

RESUMEN

6-Nitrochrysene (6-NC) is a potent mutagen in bacteria and carcinogenic in animals. It is the most potent carcinogen ever tested in newborn mouse assay. DNA lesions resulting from 6-NC modification are likely to induce mutations if they are not removed by cellular defense pathways prior to DNA replication. Earlier studies showed that 6-NC-derived C8-2'-deoxyadenosine adduct, N-(dA-8-yl)-6-AC, is very slowly repaired in human cells. In this study, we have investigated replication of N-(dA-8-yl)-6-AC in human embryonic kidney (HEK 293T) cells and the roles of translesion synthesis (TLS) DNA polymerases in bypassing it. Replication of a plasmid containing a single site-specific N-(dA-8-yl)-6-AC adduct in HEK 293 T cells showed that human DNA polymerase (hPol) η and hPol κ played important roles in bypassing the adduct, since TLS efficiency was reduced to 26 % in the absence of these two polymerases compared to 83 % in polymerase-competent HEK 293T cells. The progeny from HEK 293T cells provided 12.7 % mutants predominantly containing A→T transversions. Mutation frequency (MF) was increased to 17.8 % in hPol η-deficient cells, whereas it was decreased to 3.3 % and 3.9 % when the adduct containing plasmid was replicated in hPol κ- and hPol ζ-deficient cells, respectively. The greatest reduction in MF by more than 90 % (to MF 1.2 %) was observed in hPol ζ-knockout cells in which hPol κ was knocked down. Taken together, these results suggest that hPol κ and hPol ζ are involved in the error-prone TLS of N-(dA-8-yl)-6-AC, while hPol η performs error-free bypass.


Asunto(s)
Crisenos/química , Aductos de ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxiadenosinas/química , Aductos de ADN/química , Replicación del ADN , Células HEK293 , Humanos
3.
Environ Geochem Health ; 42(8): 2485-2494, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31264041

RESUMEN

Polycyclic aromatic hydrocarbons (PAHs) have been a major concern because of their carcinogenicity, mutagenicity, teratogenicity and wide distribution in the environment. Over 90% of PAHs in the environment exist on soil surface/sediment. Benzo[a]pyrene (BaP) is one of the predominant PAHs in soil. Thus, it is critically important to understand the patterns of BaP accumulation and transformation peculiarities in soil for the risk assessment. The studies were conducted in model experiment with Haplic Chernozem spiked with various doses of BaP (20, 200, 400 and 800 µg kg-1) equivalent to 1, 10, 20 and 40 levels of maximum permissible concentrations. The unique properties of Haplic Chernozem were studied allow to accumulate and transform BaP as well as barley plants ability to absorb of some BaP concentration. Extraction of BaP from the soil was carried out by the saponification method. The qualitative and quantitative determination of BaP and other polycyclic aromatic hydrocarbons (PAHs) was performed by high-performance liquid chromatography with fluorescence detection (Agilent 1260 Germany, 2014). BaP accumulation in soil depended on the applied BaP concentrations in Haplic Chernozem. Studying the features of PAHs transformation in the soil of a model experiment 1 year after the compound application showed the BaP content in the soil decreased up to 11-40%. Two years after the BaP application the content in the soil decreased up to 15-44% from the initial BaP content in the soil. The percentage of BaP concentration reduction in Haplic Chernozem increased with an increase in the dose of the applied xenobiotic. An increase in the dose of the applied pollutant to the soil of the model experiment contributed to an increase in all PAHs, which indicated a rapid BaP transformation in Haplic Chernozem. The PAHs content in the soils of model experiment in the first year of the research formed the following descending series: pyrene > chrysene > fluoranthene > phenanthrene. In the second year of research the phenanthrene content became higher than the fluoranthene content. The content of these compounds exceeded 20% of the total PAHs content in the soil samples in the first and second years of the model experiment. The features of PAHs accumulation and transformation in soils under artificial pollution showed the degradation of large-nuclear PAHs, starting from 5-ring polyarenes, and their structural reorganization into the less-nuclear polyarenes, such as 4-, 3-, and 2-ring PAHs. During the 2 years of the model experiment the BaP concentration in the soil decreased up to 15-44% from the initial BaP content in the soil.


Asunto(s)
Benzo(a)pireno/metabolismo , Contaminantes del Suelo/metabolismo , Benzo(a)pireno/química , Biodegradación Ambiental , Crisenos/química , Crisenos/metabolismo , Fluorenos/química , Fluorenos/metabolismo , Hordeum/metabolismo , Fenantrenos/química , Fenantrenos/metabolismo , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/metabolismo , Pirenos/química , Pirenos/metabolismo , Federación de Rusia , Suelo/química , Contaminantes del Suelo/química
4.
Molecules ; 24(6)2019 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-30884744

RESUMEN

The formation of polycyclic aromatic hydrocarbons (PAHs) is a strong global concern due to their harmful effects. To help the reduction of their emissions, a crucial understanding of their formation and a deep exploration of their growth mechanism is required. In the present work, the formation of benzo(a)pyrene was investigated computationally employing chrysene and benz(a)anthracene as starting materials. It was assumed a type of methyl addition/cyclization (MAC) was the valid growth mechanism in this case. Consequently, the reactions implied addition reactions, ring closures, hydrogen abstractions and intramolecular hydrogen shifts. These steps of the mechanism were computed to explore benzo(a)pyene formation. The corresponding energies of the chemical species were determined via hybrid density funcional theory (DFT), B3LYP/6-31+G(d,p) and M06-2X/6-311++G(d,p). Results showed that the two reaction routes had very similar trends energetically, the difference between the energy levels of the corresponding molecules was just 6.13 kJ/mol on average. The most stable structure was obtained in the benzo(a)anthracene pathway.


Asunto(s)
Benzo(a)Antracenos/química , Benzo(a)pireno/química , Carcinógenos/química , Hidrocarburos Policíclicos Aromáticos/química , Benzo(a)Antracenos/toxicidad , Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Crisenos/química , Humanos , Hidrógeno/química , Estructura Molecular , Hidrocarburos Policíclicos Aromáticos/toxicidad
5.
Luminescence ; 32(5): 845-854, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28058760

RESUMEN

Polyvinyl pyrrolidone (PVP) crowned chrysene nanoparticles (CHYNPs) were prepared by using a reprecipitation method. Dynamic light scattering (DLS) and scanning electron microscope (SEM) studies indicate that the monodispersed spherical nanoparticles bear a negative charge on their surfaces. The bathochromic spectral shift in the UV-visible and fluorescence spectrum of CHYNPs from chrysene (CHY) in acetone solution supports the J- type aggregation of nanoparticles. The aggregation-induced enhanced emission of CHYNPs at 486 and 522 nm decreases by increasing the concentration of the Ca2+ ion solution. It can display an ON-OFF type fluorescence response with high selectivity towards Ca2+ ions aqueous medium. Furthermore, the in situ generated PVP-CHYNPs-Ca2+ ensemble could recover the quenched fluorescence upon the addition of fluoride anions resulting in an OFF-ON type sensor. The present method has a correlation coefficient R2 = 0.988 with a detection limit of 1.22 µg/mL for Ca2+ in the aqueous medium. The fluorescence changes of PVP crowned CHYNPs upon the addition of Ca2+ and F- can be utilized as an INHIBIT logic gate at the molecular level, using Ca2+ and F- chemical inputs and the fluorescence intensity signal as output.


Asunto(s)
Calcio/análisis , Crisenos/química , Hierro/análisis , Sustancias Luminiscentes/química , Nanopartículas/química , Aniones/química , Dispersión Dinámica de Luz , Fluoruros/química , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Povidona/química , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Agua
6.
J Inorg Biochem ; 168: 55-66, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28013065

RESUMEN

This paper describes the synthesis of a trinuclear Cu(II) complex (4) containing a central 1,4,5,8,9,12-hexaazatriphenylene-hexacarboxylate (hat) core (3). Low, micromolar concentrations of the negatively charged parent ligand 3 and the neutral trinuclear complex 4 were found to photocleave negatively charged pUC19 plasmid DNA with high efficiency at neutral pH (350nm, 50min, 22°C). The interactions of complex 4 with double-helical DNA were studied in detail. Scavenger and colorimetric assays pointed to the formation of Cu(I), superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals during photocleavage reactions. UV-visible absorption, circular dichroism, DNA thermal denaturation, and fluorescence data suggested that the Cu(II) complex contacts double-stranded DNA in an external fashion. The persistent association of ligand 3 and complex 4 with Na(I) and/or other cations in aqueous solution might facilitate electrostatic DNA interactions.


Asunto(s)
Compuestos Aza/química , Compuestos Aza/farmacología , Crisenos/química , Crisenos/farmacología , Cobre/química , Cobre/farmacología , ADN/efectos de los fármacos , ADN/metabolismo , Procesos Fotoquímicos , Dicroismo Circular , Colorimetría , Peróxido de Hidrógeno/química , Estructura Molecular , Superóxidos/química
7.
Photochem Photobiol Sci ; 15(7): 928-36, 2016 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-27320009

RESUMEN

We investigated the photophysical properties of difluoroboronated ß-diketones (BF2DK) with chrysene and pyrene skeletons (ChB and PyB, respectively) in solution and in the solid state. Acetylchrysenes, as the key precursors to ChBs, were photochemically prepared from the corresponding (acetylphenyl)naphthylethenes by means of a modified photocyclization method. The absorption and emission spectra of the BF2DKs were obtained in chloroform and acetonitrile, and the quantum yields and lifetimes of the fluorescence were determined. Excimeric fluorescence from PyB was absent even in highly concentrated solution. Based on the Lippert-Mataga analysis of the absorption and fluorescence features, the photophysical properties of the ChBs were discussed in comparison with those of PyB. The fluorescence states of the studied BF2DKs are shown to be of a charge-transfer character. The fluorescence quantum yields decrease with increasing the solvent polarity due to the enhanced internal conversion process. The fluorescence quantum yields in the solid state of the studied BF2DKs were determined, and it was found that PyB is fluorescent, whereas the fluorescence quantum yields of the ChBs depend on the substituted position of the chrysene moiety.


Asunto(s)
Crisenos/química , Pirenos/química , Acetonitrilos/química , Crisenos/síntesis química , Ciclización , Pirenos/síntesis química , Teoría Cuántica , Solventes/química , Espectrometría de Fluorescencia , Difracción de Rayos X
8.
Chem Res Toxicol ; 29(6): 991-1002, 2016 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-27054409

RESUMEN

Exposure to polycyclic aromatic hydrocarbons (PAHs) is the major human health hazard associated with the Deepwater Horizon oil spill. C2-Chrysenes are representative PAHs present in crude oil and could contaminate the food chain. We describe the metabolism of a C2-chrysene regioisomer, 6-ethylchrysene (6-EC), in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. 6-EC-tetraol isomers were identified as signature metabolites of the diol-epoxide pathway. O-Monomethyl-O-monosulfonated-6-EC-catechol, its monohydroxy products, and N-acetyl-l-cysteine(NAC)-6-EC-ortho-quinone were discovered as signature metabolites of the ortho-quinone pathway. Potential dual metabolic activation of 6-EC involving the formation of bis-electrophiles, i.e., a mono-diol-epoxide and a mono-ortho-quinone within the same structure, bis-diol-epoxides, and bis-ortho-quinones was observed as well. The identification of 6-EC-tetraol, O-monomethyl-O-monosulfonated-6-EC-catechol, its monohydroxy products, and NAC-6-EC-ortho-quinone supports potential metabolic activation of 6-EC by P450 and AKR enzymes followed by metabolic detoxification of the ortho-quinone through interception of its redox cycling capability by catechol-O-methyltransferase and sulfotransferase enzymes. The tetraols and catechol conjugates could be used as biomarkers of human exposure to 6-EC resulting from oil spills.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Catecol O-Metiltransferasa/metabolismo , Crisenos/química , Crisenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Contaminación por Petróleo/análisis , Sulfotransferasas/metabolismo , Crisenos/análisis , Células Hep G2 , Humanos , Estructura Molecular , Células Tumorales Cultivadas
9.
Indian J Exp Biol ; 53(5): 256-63, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-26040022

RESUMEN

Degradation of chrysene, a four ringed highly carcinogenic polycyclic aromatic hydrocarbon (PAH) has been demonstrated by bacterial mixed culture Biorem-CGBD comprising Achromobacter xylosoxidans, Pseudomonas sp. and Sphingomonas sp., isolated from crude oil polluted saline sites at Bhavnagar coast, Gujarat, India. A full factorial Central Composite Design (CCD) using Response Surface Methodology (RSM) was applied to construct response surfaces, predicting 41.93% of maximum chrysene degradation with an experimental validation of 66.45% chrysene degradation on 15th day, using a combination of 0.175, 0.175 and 0.385 mL of OD600 = 1 inoculum of A. xylosoxidans, Pseudomonas sp. and Sphingomonas sp., respectively and a regression coefficient (R2) of 0.9485 indicating reproducibility of the experiment. It was observed that chrysene degradation can be successfully enhanced using RSM, making mixed culture Biorem-CGBD a potential bioremediation target for PAH contaminated saline sites.


Asunto(s)
Biodegradación Ambiental , Crisenos/química , Hidrocarburos Policíclicos Aromáticos/química , Achromobacter denitrificans/química , Achromobacter denitrificans/metabolismo , Carcinógenos/química , Carcinógenos/metabolismo , Crisenos/toxicidad , Humanos , Hidrocarburos Policíclicos Aromáticos/toxicidad , Pseudomonas/química , Pseudomonas/metabolismo , Sphingomonas/química , Sphingomonas/metabolismo
10.
Environ Toxicol Chem ; 33(8): 1792-801, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24764175

RESUMEN

The present study examines concentrations and risks of polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs (NPAHs), steranes, and hopanes in lake trout collected in Lake Michigan. A total of 74 fish were collected in 2 seasons at 3 offshore sites. The total PAH concentration (Σ9 PAH) in whole fish ranged from 223 pg/g to 1704 pg/g wet weight, and PAH concentrations and profiles were similar across season, site, and sex. The total NPAH (Σ9 NPAH) concentrations ranged from 0.2 pg/g to 31 pg/g wet weight, and carcinogenic compounds, including 1-nitropyrene and 6-nitrochrysene, were detected. In the fall, NPAH concentrations were low at the Illinois site (0.2-0.5 pg/g wet wt), and site profiles differed considerably; in the spring, concentrations and profiles were similar across sites, possibly reflecting changes in fish behavior. In the fall, the total sterane (Σ5 Sterane) and total hopane (Σ2 Hopane) levels reached 808 pg/g and 141 pg/g wet weight, respectively, but concentrations in the spring were 10 times lower. Concentrations in eggs (fall only) were on the same order of magnitude as those in whole fish. These results demonstrate the presence of target semivolatile organic compounds in a top predator fish, and are consistent with PAH biodilution observed previously. Using the available toxicity information for PAHs and NPAHs, the expected cancer risk from consumption of lake trout sampled are low. However, NPAHs contributed a significant portion of the toxic equivalencies in some samples. The present study provides the first measurements of NPAHs in freshwater fish, and results suggest that additional assessment is warranted.


Asunto(s)
Monitoreo del Ambiente/métodos , Hidrocarburos Aromáticos/análisis , Lagos/química , Trucha , Contaminantes Químicos del Agua/análisis , Animales , Biota , Crisenos/análisis , Crisenos/química , Crisenos/toxicidad , Humanos , Hidrocarburos Aromáticos/química , Hidrocarburos Aromáticos/toxicidad , Illinois , Fenantrenos/análisis , Fenantrenos/química , Fenantrenos/toxicidad , Hidrocarburos Policíclicos Aromáticos/análisis , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/toxicidad , Pirenos/análisis , Pirenos/química , Pirenos/toxicidad , Triterpenos/análisis , Triterpenos/química , Triterpenos/toxicidad , Volatilización , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/toxicidad
11.
PLoS One ; 9(3): e89668, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24598537

RESUMEN

Enterovirus 71 (EV71) can cause severe disease and even lead to death in children, and an effective antiviral drug is currently unavailable. The anti-EV71 effect of chrysin (5,7-dihydroxyflavone), a natural flavonoid commonly found in many plants, was tested in this report. By using the predicting program Autodock 4.0 and an in vitro protease inhibition assay, we found that chrysin could suppress viral 3Cpro activity. Replication of viral RNA and production of viral capsid protein and the infectious virion were strongly inhibited by chrysin, without noticeable cytotoxicity. Cytopathic effects on cells were also prevented. Diisopropyl chrysin-7-yl phosphate (CPI), the phosphate ester for chrysin, was generated through a simplified Atheron-Todd reaction to achieve stronger anti-viral activity. CPI was also able to bind with and inhibit viral 3Cpro activity in vitro. As expected, CPI demonstrated more potent antiviral activity against EV71.


Asunto(s)
Antivirales/farmacología , Crisenos/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Enterovirus Humano A/fisiología , Flavonoides/farmacología , Organofosfatos/farmacología , Proteasas Virales 3C , Antivirales/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Crisenos/química , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/química , Evaluación Preclínica de Medicamentos , Enterovirus Humano A/efectos de los fármacos , Flavonoides/química , Humanos , Simulación del Acoplamiento Molecular , Organofosfatos/química , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Replicación Viral/efectos de los fármacos
12.
Chem Res Toxicol ; 26(11): 1746-54, 2013 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-24112095

RESUMEN

Previous studies in rats, mice, and in vitro systems showed that 6-NC can be metabolically activated by two major pathways: (1) the formation of N-hydroxy-6-aminochrysene by nitroreduction to yield three major adducts, N-(dG-8-yl)-6-AC, 5-(dG-N(2)-yl)-6-AC, and N-(dA-8-yl)-6-AC, and (2) the formation of trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C) by a combination of nitroreduction and ring oxidation pathways to yield N-(dG-8-yl)-1,2-DHD-6-AC, 5-(dG-N(2)-yl)-1,2-DHD-6-AC and N-(dA-8-yl)-1,2-DHD-6-AC. These DNA lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. Here, we compared for the first time, in HeLa cell extracts in vitro, the relative nucleotide excision repair (NER) efficiencies of DNA lesions derived from simple nitroreduction and from a combination of nitroreduction and ring oxidation pathways. We show that the N-(dG-8-yl)-1,2-DHD-6-AC adduct is more resistant to NER than the N-(dG-8-yl)-6-AC adduct by a factor of ∼2. Furthermore, the N-(dA-8-yl)-6-AC is much more resistant to repair since its NER efficiency is ∼8-fold lower than that of the N-(dG-8-yl)-6-AC adduct. On the basis of our previous study and the present investigation, lesions derived from 6-NC and benzo[a]pyrene can be ranked from the most to the least resistant lesion as follows: N-(dA-8-yl)-6-AC > N-(dG-8-yl)-1,2-DHD-6-AC > 5-(dG-N(2)-yl)-6-AC ≃ N-(dG-8-yl)-6-AC ≃ (+)-7R,8S,9S,10S-benzo[a]pyrene diol epoxide-derived trans-anti-benzo[a]pyrene-N(2)-dG adduct. The slow repair of the various lesions derived from 6-NC and thus their potential persistence in mammalian tissue could in part account for the powerful carcinogenicity of 6-NC as compared to B[a]P in the rat mammary gland.


Asunto(s)
Adenina/química , Crisenos/química , Aductos de ADN/metabolismo , Reparación del ADN , Guanina/química , Animales , Benzo(a)pireno/química , Bovinos , ADN/química , ADN/metabolismo , Aductos de ADN/análisis , Aductos de ADN/química , Células HeLa , Humanos , Ratones , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Oxidación-Reducción , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
13.
Carcinogenesis ; 34(9): 2184-91, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23671133

RESUMEN

Each enantiomer of the diastereomeric pair of bay-region dibenz[a,h]anthracene 3,4-diol-1,2-epoxides in which the benzylic 4-hydroxyl group and epoxide oxygen are either cis (isomer 1) or trans (isomer 2) were evaluated for mutagenic activity. In strains TA 98 and TA 100 of Salmonella typhimurium, the diol epoxide with (1S,2R,3S,4R) absolute configuration [(-)-diol epoxide-1] had the highest mutagenic activity. In Chinese hamster V-79 cells, the diol epoxide with (1R,2S,3S,4R) absolute configuration [(+)-diol epoxide-2] had the highest mutagenic activity. The (1R,2S,3R,4S) diol epoxide [(+)-diol epoxide-1] also had appreciable activity, whereas the other two bay-region diol epoxide enantiomers had very low activity. In tumor studies, the (1R,2S,3S,4R) enantiomer was the only diol epoxide isomer tested that had strong activity as a tumor initiator on mouse skin and in causing lung and liver tumors when injected into newborn mice. This stereoisomer was about one-third as active as the parent hydrocarbon, dibenz[a,h]anthracene as a tumor initiator on mouse skin; it was several-fold more active than dibenz[a,h]anthracene as a lung and liver carcinogen when injected into newborn mice. (-)-(3R,4R)-3ß,4α-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(-)-3,4-dihydrodiol] was slightly more active than dibenz[a,h]anthracene as a tumor initiator on mouse skin, whereas (+)-(3S,4S)-3α,4ß-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(+)-3,4-dihydrodiol] had only very weak activity. The present investigation and previous studies with the corresponding four possible enantiopure bay-region diol epoxide enantiomers/diastereomers of benzo[a]pyrene, benz[a]anthracene, chrysene, benzo[c]phenanthrene, dibenz[c,h]acridine, dibenz[a,h]acridine and dibenz[a,h]anthracene indicate that the bay-region diol epoxide enantiomer with [R,S,S,R] absolute stereochemistry has high tumorigenic activity on mouse skin and in newborn mice.


Asunto(s)
Carcinogénesis/patología , Crisenos/farmacología , Compuestos Epoxi/farmacología , Neoplasias Cutáneas/inducido químicamente , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/química , Crisenos/química , Crisenos/toxicidad , Cricetinae , Compuestos Epoxi/toxicidad , Humanos , Ratones , Mutagénesis/efectos de los fármacos , Mutágenos/farmacología , Mutágenos/toxicidad , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Neoplasias Cutáneas/patología , Estereoisomerismo , Relación Estructura-Actividad
14.
Bioelectrochemistry ; 92: 42-6, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23603150

RESUMEN

The patch clamp technique in the whole cell configuration is potentially a powerful tool to investigate electroporation (electric-field-induced membrane permeabilization). Membrane polarization beyond certain threshold voltages leads to a steep conductance increase either indicating field-induced pore formation or being due to patch clamp artifacts (seal resistance breakdown). Protoplasts derived from tobacco culture cell lines (Bright Yellow-2, BY-2; Virginia bright Italian-0, VBI-0) were stained with the voltage-sensitive dye ANNINE-6. After establishing the whole cell patch clamp configuration 50-ms command voltage (Ucomm) steps ranging from -500 mV to +500 mV were applied while simultaneously exposing protoplasts to light at 475 nm wavelength. Pulse-induced currents and fluorescence intensity (known to be linearly related to the trans-membrane voltage, Um) were recorded. Plotting fluorescence intensity against Ucomm revealed saturation of the curve at values<-300 mV and >+300 mV and close correlation with theoretical Um values calculated on the basis of membrane pore formation. For BY-2 and VBI-0 protoplasts ANNINE-6 voltage sensitivity was calculated to be -0.0014 mV(-1) and -0.0012 mV(-1), respectively. Voltage ramp experiments revealed cation-selectivity of field-induced pores. Anions are conducted poorly independent of their size. In conclusion, the patch clamp technique is validated as a useful tool in electroporation research.


Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Crisenos/química , Electroporación/métodos , Colorantes Fluorescentes/química , Técnicas de Placa-Clamp/métodos , Compuestos de Amonio Cuaternario/química , Microscopía Fluorescente , Protoplastos/citología , Nicotiana/citología , Imagen de Colorante Sensible al Voltaje
15.
J Inorg Biochem ; 120: 1-7, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23262457

RESUMEN

Chrysene and pyrene are known toxic compounds recalcitrant to biodegradation. Here directed evolution allowed us to identify two new mutants of cytochrome P450 BM3 that are able to hydroxylate both compounds. Random mutagenesis has been used to generate libraries of mutants of P450 BM3 active toward polycyclic aromatic hydrocarbons (PAHs) PAHs. After two rounds of error-prone PCR and backcross with parental DNA, three mutants were identified for improved activity toward pyrene and for the first time a new activity toward chrysene in comparison to the wild type enzyme. The mutants show higher affinity and coupling efficiency for chrysene with faster rates of product formation compared to the wild type. Furthermore, the mutants are able to hydroxylate chrysene in different positions, producing four metabolites, 1-, 3-, 4-, and 6-hydroxychrysene, and to hydroxylate pyrene to 1-hydroxypyrene. The majority of the mutation sites are found to be far from the active site, demonstrating the power of directed evolution in identifying mutations difficult to predict with a rational design approach. The different product profiles obtained for the different P450 BM3 mutants indicate that substrate orientation in the catalytic pocket of the protein can be modified by protein engineering. The mutants can be used for metabolic engineering for safe and cost-effective sustainable production of hydroxylated PAHs for industrial purposes as well as for the assessment of their carcinogenic activity in mammals.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Evolución Molecular Dirigida/métodos , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Crisenos/análisis , Crisenos/química , Crisenos/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Cromatografía de Gases y Espectrometría de Masas , Hidroxilación , NADP/química , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/aislamiento & purificación , Oxidación-Reducción , Hidrocarburos Policíclicos Aromáticos/química , Conformación Proteica , Ingeniería de Proteínas , Pirenos/química , Pirenos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación
16.
ScientificWorldJournal ; 2012: 828343, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23346024

RESUMEN

Polycyclic aromatic hydrocarbons (PAHs) are a concern due to their carcinogenicity and propensity for transboundary atmospheric transport. Ireland is located on the western periphery of Europe and assumed to receive clean Atlantic air. As such, it has been used as an atmospheric reference for comparison to other regions. Nonetheless, few studies have evaluated concentrations of PAHs within the Irish environment. In the current study, PAHs were measured at five upland (500-800 masl) headwater lake catchments in coastal regions around Ireland, remote from industrial point source emissions. Semipermeable membrane devices were deployed in lakes for a 6-month period in July 2009, and topsoils were sampled from each catchment during October 2010. The concentrations of PAHs were low at most study sites with respect to other temperate regions. Homologue groups partitioned between lake and soil compartments based on their molecular weight were: "lighter" substances, such as Phenanthrene and Fluorene, were found in higher proportions in lakes, whereas "heavier" compounds, such as Chrysene and Benz[a]anthracene, were more prominent in soils. Concentrations of PAHs were highest at the east coast sites, potentially due to contributions from historical transboundary and regional combustion sources.


Asunto(s)
Lagos/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Contaminantes del Suelo/análisis , Contaminantes Químicos del Agua/análisis , Benzo(a)Antracenos/análisis , Benzo(a)Antracenos/química , Crisenos/análisis , Crisenos/química , Monitoreo del Ambiente/métodos , Fluorenos/análisis , Fluorenos/química , Cromatografía de Gases y Espectrometría de Masas , Geografía , Sedimentos Geológicos/análisis , Sedimentos Geológicos/química , Irlanda , Lagos/química , Peso Molecular , Fenantrenos/análisis , Fenantrenos/química , Hidrocarburos Policíclicos Aromáticos/química , Análisis de Componente Principal , Suelo/análisis , Suelo/química , Movimientos del Agua
17.
Rep Carcinog ; 12: 353-61, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21863085
19.
Chem Res Toxicol ; 24(9): 1549-59, 2011 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-21780761

RESUMEN

Dibenzo[a,l]pyrene (DB[a,l]P) (dibenzo[def,p]chrysene) is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) that has been identified in tobacco smoke and is found in our environment due to incomplete combustion of organic matter. Its metabolites are known to form stable DNA adducts in bacteria and mammalian cells, and can lead to tumors in animal models. Glucuronidation of major metabolites of DB[a,l]P by the uridine-5'-diphosphate glucuronosyltransferase (UGT) family of enzymes is an important route of detoxification of this pro-carcinogen. The focus of the current study was to characterize the glucuronidation of the pro-carcinogenic enantiomers DB[a,l]P-(+)-trans-11S,12S-diol and DB[a,l]P-(-)-trans-11R,12R-diol. Glucuronidation assays with HEK293 cell lines overexpressing individual human UGT enzymes demonstrated that UGTs 1A1, 1A4, 1A7, 1A8, 1A9, 1A10, and 2B7 glucuronidated one or both DB[a,l]P-trans-11,12-diol enantiomers. Three glucuronide conjugates were observed in activity assays with UGTs 1A1 and 1A10, while two glucuronides were formed by UGTs 1A7, 1A8, and 1A9, and one glucuronide was made by UGT1A4 and UGT2B7. Enzyme kinetic analysis indicated that UGT1A9 was the most efficient UGT at forming both the (+)-DB[a,l]P-11-Gluc and (-)-DB[a,l]P-11-Gluc products, while UGTs 1A1 and 1A10 were the most efficient at forming the (+)-DB[a,l]P-12-Gluc product (as determined by k(cat)/K(M)). Incubations with human liver microsomes showed the formation of three diastereomeric glucuronide products: (+)-DB[a,l]P-11-Gluc, (+)-DB[a,l]P-12-Gluc, and (-)-DB[a,l]P-11-Gluc, with an average overall ratio of 31:32:37 in four liver specimens. Human bronchus and trachea tissue homogenates demonstrated glucuronidation activity against both DB[a,l]P-trans-11,12-diol enantiomers, with both tissues producing the (+)-DB[a,l]P-11-Gluc and (+)-DB[a,l]P-12-Gluc with little or no formation of (-)-DB[a,l]P-11-Gluc. These results indicate that multiple UGTs are involved in the stereospecific glucuronidation of DB[a,l]P-trans-11,12-diol in a pattern consistent with their expression in respiratory tract tissues and that glucuronidation may be an important first-line detoxification mechanism of DB[a,l]P metabolites.


Asunto(s)
Carcinógenos/metabolismo , Crisenos/metabolismo , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Bronquios/metabolismo , Carcinógenos/química , Línea Celular , Crisenos/química , Glucurónidos/química , Humanos , Estereoisomerismo , Tráquea/metabolismo
20.
Chem Res Toxicol ; 24(1): 65-72, 2011 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-21114286

RESUMEN

Ubiquitous environmental agents [e.g., polynuclear aromatic hydrocarbons (PAHs) and their nitrated derivatives (NO(2)-PAHs)] that are known to induce mammary cancer in rodents are regarded as potential human risk factors for inducing analogous human cancers. Although 6-nitrochrysene (6-NC) is less abundant than other NO(2)-PAHs in the environment, it is the most potent mammary carcinogen in the rat; its carcinogenic potency is not only higher than that of the carcinogenic PAH, benzo[a]pyrene (B[a]P), but also of the well-known carcinogenic heterocylic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). Studies in rats and in vitro assays have indicated that 6-NC can be activated by simple nitroreduction leading to the formation of 6-hydroxylaminochrysene (N-OH-6-AC); this metabolite yielded N-(deoxyguanosin-8-yl)-6-aminochrysene (N-[dG-8-yl]-6-AC) and 5-(deoxyguanosin-N(2)-yl)-6-aminochrysene (5-[dG-N(2)-yl]-6-AC. These lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. However, nothing is known about the susceptibility of these adducts to nucleotide excision repair (NER), the major cellular repair system that removes bulky adducts. In order to address this issue, we synthesized the N-(dG-8-yl)-6-AC and 5-(dG- N(2)-yl)-6-AC lesions and site-specifically inserted these lesions into 135-mer DNA duplexes. These constructs were incubated with NER-competent nuclear extracts from human HeLa cells. The efficiency of repair of these lesions was ∼ 8 times less efficient than that in the case of the well-known and excellent substrate of NER, the intrastrand cross-linked cis-diaminodichloroplatinum II adduct in double-stranded DNA (cis-Pt), but similar to N(2)-dG adducts derived from the (+)-bay region diol epoxide of B[a]P [(+)-trans-B[a]P-N(2)-dG]. The results support the hypothesis that the N-(dG-8-yl)-6-AC and 5-(dG-N(2)-yl)-6-AC lesions may be slowly repaired and thus persistent in mammalian tissue which could, in part, account for the potent tumorigenic activity of 6-NC in the rat mammary gland.


Asunto(s)
Crisenos/química , Aductos de ADN/química , Reparación del ADN , Desoxiguanosina/análogos & derivados , Animales , Benzo(a)pireno/química , Benzo(a)pireno/toxicidad , Cromatografía Líquida de Alta Presión , Crisenos/toxicidad , Desoxiguanosina/química , Células HeLa , Humanos , Imidazoles/química , Imidazoles/toxicidad , Espectrometría de Masas , Oligonucleótidos/metabolismo , Ratas
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