RESUMO
The well known biomarker of oxidative stress, 8-oxo-7,8-dihydroguanine, is more susceptible to further oxidation than the parent guanine base and can be oxidatively transformed to the genotoxic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) lesions. Incubation of 135-mer duplexes with single Sp or Gh lesions in human cell extracts yields a characteristic nucleotide excision repair (NER)-induced ladder of short dual incision oligonucleotide fragments in addition to base excision repair (BER) incision products. The ladders were not observed when NER was inhibited either by mouse monoclonal antibody (5F12) to human XPA or in XPC(-/-) fibroblast cell extracts. However, normal NER activity appeared when the XPC(-/-) cell extracts were complemented with XPC-RAD23B proteins. The Sp and Gh lesions are excellent substrates of both BER and NER. In contrast, 5-guanidino-4-nitroimidazole, a product of the oxidation of guanine in DNA by peroxynitrite, is an excellent substrate of BER only. In the case of mouse embryonic fibroblasts, BER of the Sp lesion is strongly reduced in NEIL1(-/-) relative to NEIL1(+/+) extracts. In summary, in human cell extracts, BER and NER activities co-exist and excise Gh and Sp DNA lesions, suggesting that the relative NER/BER product ratios may depend on competitive BER and NER protein binding to these lesions.
Assuntos
Reparo do DNA , Guanina/análogos & derivados , Estresse Oxidativo , Animais , Linhagem Celular , Células , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Guanina/metabolismo , Guanina/toxicidade , Células HeLa , Humanos , CamundongosRESUMO
Hospital- and community-acquired, complicated skin and soft tissue infections, often attributed to Staphylococcus aureus and Streptococcus pyogenes, present a significant health burden that is associated with increased health care costs and mortality. As these two species are difficult to discern on diagnosis and are associated with differential profiles of drug resistance, the development of an efficacious antibacterial agent that targets both organisms is a high priority. Herein we describe a structure-based drug development effort that has produced highly potent inhibitors of dihydrofolate reductase from both species. Optimized propargyl-linked antifolates containing a key pyridyl substituent display antibacterial activity against both methicillin-resistant S. aureus and S. pyogenes at MIC values below 0.1 µg/mL and minimal cytotoxicity against mammalian cells. Further evaluation against a panel of clinical isolates shows good efficacy against a range of important phenotypes such as hospital- and community-acquired strains as well as strains resistant to vancomycin.
Assuntos
Antagonistas do Ácido Fólico/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Dermatopatias/tratamento farmacológico , Infecções dos Tecidos Moles/tratamento farmacológico , Streptococcus pyogenes/efeitos dos fármacos , Alcinos/uso terapêutico , Antibacterianos , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Dermatopatias/microbiologia , Infecções dos Tecidos Moles/microbiologia , Especificidade da Espécie , Vancomicina/farmacologiaRESUMO
Chromate is a human carcinogen with a poorly defined mechanism of DNA damage. In vitro and prokaryotic studies have shown that DNA damage may occur via the formation of the hydantoin lesions guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) from further oxidation of 8-oxo-7,8-dihydroguanine (8oxoG). The unusual structure of these lesions coupled with their enhanced mutagenicity make them attractive for study with regard to their role in chromate-induced cancer. We have studied the formation of Gh versus Sp and their associated diastereomers following oxidation by model Cr(V) complexes and from in situ chromate reduction by ascorbate and glutathione. Identification of the two optically assigned diastereomers of Sp (R-Sp and S-Sp) as well as the two diastereomers of Gh (Gh1 and Gh2, not yet optically assigned) was carried out using increasingly sterically hindered substrates (nucleoside --> ssDNA --> dsDNA). Lesion formation and diastereomeric preference were found to be highly oxidant- and substrate-dependent. The Ir(IV)-positive control showed a shift from near equal levels of Gh and Sp and near equal levels of all four diastereomers in the nucleoside to all Gh formation in dsDNA, with a 5-fold enhancement in Gh2 over Gh1. The two model Cr(V) complexes used in this study, Cr(V)-salen and Cr(V)-ehba, showed opposite trends going from nucleoside to dsDNA with Cr(V)-salen giving enhanced Sp formation (with mainly R-Sp formed) and the Cr(V)-ehba having an oxidation profile nearly identical to that of Ir(IV). The two chromate reduction systems, Cr(6+)/ascorbate and Cr(6+)/glutathione, designed to model the intracellular reduction of chromate, showed lower levels of oxidation in all substrates. Notable in this group was the shift in the formation of the lesions to essentially all Sp for the Cr(6+)/ascorbate system with the most sterically hindered substrate, dsDNA. These results, when coupled with the known diastereomeric preference for excision of hydantoin lesions by the hNEIL1 enzyme, show the importance of defining both levels of lesion formation and diastereomeric preference of formation with regard to their potential impact on chromate carcinogenesis.
Assuntos
Cromatos/metabolismo , Guanidinas/química , Guanosina/análogos & derivados , Hidantoínas/química , Compostos de Espiro/química , Cromatos/química , Guanosina/química , Humanos , Modelos Moleculares , Oxirredução , Estresse Oxidativo , Espectrometria de Massas por Ionização por Electrospray , EstereoisomerismoRESUMO
Inorganic arsenic that is ingested through drinking water or inhalation is metabolized by biological methylation pathways into organoarsenical metabolites. It is now becoming understood that this metabolism that was formerly considered to be detoxification may contribute as much or more to increasing the toxicity of arsenic. One proposed mode of the toxic action of arsenic and its organoarsenic metabolites is through its binding to proteins and inactivating their enzymatic activity. The classic case has been considered the affinity of the proximal 1,3 sulfhydryl groups of the lipoic acid cofactor of the pyruvate dehydrogenase complex for arsenic. A 2:1 stoichiometry of sulfhydryl to arsenic groups has been measured in proteins and arsenical complexes can be synthesized using free D,L-lipoic acid. The relative importance of this site for arsenic binding has come in to question through the use of methylating bifunctional arsenic complexes that suggested the methylation of an active site histidine may also be important, and the suggestion that arsenic inhibits the pyruvate dehydrogenase complex indirectly by elevating mitochondrial hydrogen peroxide generation. In order to separate the effects of direct trivalent arsenite toxicity from that of hydrogen peroxide and activated oxygen, we studied the inhibition of the PDH complex under conditions that did not generate hydrogen peroxide but did expose the lipoic acid group in its reduced state to arsenicals. We also studied the effects of arsenicals in the inhibition of the α-ketoglutarate dehydrogenase complex. We found that only trivalent arsenical compounds inhibited the activity of both dehydrogenase complexes and only when the lipoic acid was in its reduced form. Arsenite inhibited both enzyme complexes approximately equivalently while monomethylarsenite inhibited the PDH complex to a greater extent than the KGDH complex - although both complexes were very sensitive to inhibition by this complex. Dimethylarsenite inhibition of both complexes was only observed with longer pre-incubation periods. Cumulative inhibition by the reduced arsenical was observed for all complexes indicating a binding mode of inhibition that is dependent upon lipoic acid being in its reduced state.
RESUMO
The human A549 lung cell line is used in this study as a model to evaluate chromium toxicity and mutagenesis since inhalation exposure of this metal gives rise to an epidemiology that indicates the lung as a target organ of chromium toxicity. Hexavalent chromium is considered the carcinogenic form of chromium, however it must be reductively activated following uptake into cells in order to react with intracellular constituents. We have previously established that the fluorescent dyes, dichlorofluorescein (DCF) and dihydrorhodamine, are effective indicators of the reductive activation of chromium and are sensitive measures of the formation of highly reactive chromium species (RCS) intracellularly. In order to examine the role of the two common intracellular reductants, glutathione and ascorbic acid (Vitamin C) in generating RCS intracellularly, we manipulated their intracellular levels through the use of buthionine sulfoximine (BSO) or by the addition of ascorbate into the culture media. We found that the high levels of glutathione in this cancer cell line lowered endogenous oxidation levels markedly, and that, by decreasing intracellular glutathione, BSO not only generated a higher background level of endogenous intracellular oxidation but the chromium-stimulated oxidation also increased markedly. Contrary to it appellation as an anti-oxidant, ascorbic acid stimulated a strong pro-oxidant response upon chromium treatment and this pro-oxidant response was evident regardless of the levels of glutathione in the cells. Based on these results, we conclude that ascorbic acid acts as a pro-oxidant in chromium-treated cells.
Assuntos
Ácido Ascórbico/metabolismo , Compostos de Cromo/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ácido Ascórbico/química , Ácido Ascórbico/farmacologia , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Compostos de Cromo/química , Relação Dose-Resposta a Droga , Fluorescência , Glutationa/metabolismo , Humanos , Modelos Químicos , Estrutura Molecular , Oxirredução/efeitos dos fármacosRESUMO
Growth inhibition and oxidized guanine lesion formation were studied in a number of base excision repair (BER) deficient Escherichia coli (E. coli) following chromate exposure. The only BER deficient bacterial strain that demonstrated significant growth inhibition by chromate, in comparison to its matched wild-type cell line, was the Nei deficient (TK3D11). HPLC coupled with electrospray ionization mass spectrometry showed that the Nei deficient E. coli accumulated the further oxidized guanine lesion, spiroiminodihydantoin (Sp), in genomic DNA at levels that were approximately 20-fold greater than its wild-type counterpart. However, no accumulation of the putative intermediate of Sp, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), was observed in the Nei deficient strain. A MutM-/MutY- double deletion mutant that was deficient in BER enzymes for the recognition and repair of 8-oxodG demonstrated no sensitivity toward chromate nor was there an associated increase in Sp accumulation over that of its wild type. However, the MutM-/MutY- double deletion mutant did show approximately 20-fold accumulation of 8-oxodG upon chromate exposure over that of the wild type and the Nei deficient E. coli. These data demonstrate that the Nei BER enzyme is critical for the recognition and repair of the Sp lesion in bacterial cell lines and demonstrates the protective effect of a specific BER enzyme on DNA lesions formed by chromate. To our knowledge, these are the first studies to show the formation and biological significance of the Sp lesion in a cellular system. This study has significant mechanistic and toxicological implications for how chromate may serve as an initiator of carcinogenesis and suggests a role for specific repair enzymes that may ameliorate the carcinogenic potential of chromate.
Assuntos
Cromatos/farmacologia , Desoxirribonuclease (Dímero de Pirimidina)/deficiência , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Guanina/metabolismo , Guanosina/análogos & derivados , Compostos de Espiro/metabolismo , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Desoxirribonuclease (Dímero de Pirimidina)/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genoma Bacteriano/genética , Guanina/química , Guanosina/química , Guanosina/metabolismo , Estrutura Molecular , Oxirredução , Espectrometria de Massas por Ionização por Electrospray , Compostos de Espiro/químicaRESUMO
7,8-dihydro-8-oxoguanine (8-oxoG) is thought to be a major lesion formed in DNA by oxidative attack at the nucleobase guanine. Recent studies have shown that 8-oxoG has a lower reduction potential than the parent guanine and is a hot spot for further oxidation. Spiroiminodihydantoin (Sp) has been identified as one of these further oxidation products. Chromium(VI) is a human carcinogen that, when reduced by a cellular reductant such as ascorbate, can oxidize DNA. In this study, duplex DNA was reacted with Cr(VI) and ascorbate to identify and quantify the base lesions formed. Guanine bases were observed to be preferentially oxidized with 5' guanines within purine repeats showing enhanced oxidation. Trapping of the guanine lesions by the base excision repair enzymes hOGG1 and mNEIL2 showed nearly exclusive trapping by mNEIL2, suggesting that 8-oxoG was not the major lesion but rather a lesion recognized by mNEIL2 such as Sp. Formation of the Sp lesion in the Cr(VI)/Asc oxidation reaction with DNA was confirmed by LC-ESI-MS detection. HPLC-ECD was used to identify and quantify any 8-oxoG arising from Cr(VI)/Asc oxidation of DNA. Concentrations of Cr(VI) (3.1-50 microM) with a corresponding 1:10 ratio of Asc oxidized between 0.3% and 1.5% of all guanines within the duplex DNA strand to Sp. 8-oxoG was also identified but with the highest Cr(VI) concentration converting approximately 0.1% of all guanines to 8-oxoG. These results show that Sp was present in concentrations approximately 20 times greater than that of 8-oxoG in this system. The results indicate that 8-oxoG, while present, was not the major product of Cr(VI)/Asc oxidation of DNA and that Sp predominates under these conditions. These results further imply that Sp may be the lesion that accounts for the carcinogenicity of this metal in cellular systems.
Assuntos
Ácido Ascórbico/química , Cromo/química , DNA/química , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Guanina/metabolismo , Guanosina/análogos & derivados , Compostos de Espiro/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Ácido Ascórbico/farmacologia , Cromo/farmacologia , DNA Glicosilases/metabolismo , Reparo do DNA , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Guanina/química , Guanosina/química , Guanosina/metabolismo , Estrutura Molecular , Oxirredução/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray , Compostos de Espiro/químicaRESUMO
8-Oxoguanine (8-oxoG) is an unstable mutagenic DNA lesion that is prone to further oxidation. High valent metals such as Cr(V) and Ir(IV) readily oxidize 8-oxoG to form guanidinohydantoin (Gh), its isomer iminoallantoin (Ia), and spiroiminodihydantoin (Sp). When present in DNA, these lesions show enhanced base misincorporation over the parent 8-oxoG lesion leading to G --> T and G --> C transversion mutations and polymerase arrest. These findings suggested that further oxidized lesions of 8-oxoG are more mutagenic and toxic than 8-oxoG itself. Repair of oxidatively damaged bases, including Sp and Gh/Ia, are initiated by the base excision repair (BER) system that involves the DNA glycosylases Fpg, Nei, and Nth in E. coli. Mammalian homologs of two of these BER enzymes, OGG1 and NTH1, have little or no affinity for Gh/Ia and Sp. Herein we report that two recently identified mammalian glycosylases, NEIL1 and NEIL2, showed a high affinity for recognition and cleavage of DNA containing Gh/Ia and Sp lesions. NEIL1 and NEIL2 recognized both of these lesions in single-stranded DNA and catalyzed the removal of the lesions through a beta- and delta-elimination mechanism. NEIL1 and NEIL2 also recognized and excised the Gh/Ia lesion opposite all four natural bases in double-stranded DNA. NEIL1 was able to excise the Sp lesion opposite the four natural bases in double-stranded DNA, however, NEIL2 showed little cleavage activity against the Sp lesion in duplex DNA although DNA trapping studies show recognition and binding of NEIL2 to this lesion. This work suggests that NEIL1 and NEIL2 are essential in the recognition of further oxidized lesions arising from 8-oxoG and implies that these BER glycosylases may play an important role in the repair of DNA damage induced by carcinogenic metals.
Assuntos
Dano ao DNA/genética , DNA Glicosilases/metabolismo , Reparo do DNA/genética , Guanidinas/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Hidantoínas/metabolismo , Compostos de Espiro/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Compostos de Cromo/química , Compostos de Cromo/metabolismo , Clonagem Molecular , DNA Glicosilases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Guanosina/química , Camundongos , OligonucleotídeosRESUMO
Human exposure to toxic metals and metalloids in the environment seldom occurs from a single pure compound. Most environmental exposure profiles are heterogeneous with co-exposure occurring coincident with multiple toxic metal species. This co-exposure to metals and metalloids in complex mixtures can result in a synergistic, additive or even depletive toxic response. The complexity of interactions presented by metal mixtures presents a need for convenient and sensitive methods to determine potential toxic responses from such co-exposure. We have studied the reaction between the two commonly associated toxic metals of chromate, Cr(VI), and arsenite, As(III), with regards to the ability of As(III) to reductively activate Cr(VI) to generate oxidative stress and DNA damage. Using a DCF-based fluorescent dye assay we have demonstrated that the redox reaction between As(III) and Cr(VI) yields high valent intermediates of chromium, Cr(V), that are highly oxidizing. This induction of oxidizing potential was dose dependent and did not occur with As(III) or Cr(VI) alone or, with the other major oxidation state of arsenic, arsenate, As(V). The mechanism of oxidation of DCFH to the fluorescent species, DCF, in this reaction was through a direct, metal-based oxidation since addition of radical scavengers did not significantly decrease oxidation of the dye in this system. The addition of a ligand that stabilizes the high valent Cr(V) oxidation state, 2-ethyl-2-hydroxybutyric acid (EHBA), to the chromate and arsenite mixture resulted in an enhancement of DCF fluorescence. The DCF fluorescence observed with the Cr(VI) and As(III) mixture was also found to correlate with oxidative DNA damage as measured by a plasmid nicking assay. These data show how metal-metal interactions in environmental mixtures could result in the synergistic induction of oxidative stress and DNA damage. Further, these data demonstrate the utility of the DCF fluorescence assay as a sensitive method for screening synergistic redox interactions in metal mixtures.
Assuntos
Arsenitos/toxicidade , Carcinógenos Ambientais/toxicidade , Cromatos/toxicidade , Cromo/toxicidade , Dano ao DNA , Modelos Teóricos , Arsenitos/química , Carcinógenos Ambientais/química , Cromatos/química , Cromo/química , Interações Medicamentosas , Fluoresceínas/química , Fluorescência , Oxirredução , Estresse OxidativoRESUMO
A number of common promoter elements that drive transcription of redox sensitive genes have runs of guanines in their transcription factor recognition sequence. A paradox exists insomuch that the same guanine runs necessary for transcription factor recognition are thermodynamically prone to oxidative modification, potentially altering the binding affinity of transcription factors. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) is a common oxidative modification of guanine that is generated by a variety of metals and reactive oxygen species. We have used the p50 subunit of the NF-kappaB transcription factor to show that oxidation of guanine to 8-oxo-dG at sites critical for protein recognition impacts transcription factor binding affinity differently depending upon the site of oxidation. It can be argued that the impact of such oxidation will be minimal in repair proficient cells. Therefore, we have developed an assay to assess the ability of these lesions to be shielded by transcription factor binding from recognition and repair by base excision repair (BER) enzymes. In this study, 8-oxo-dG was substituted for guanine at sites G(1)-G(4) in the NF-kappaB sequence 5'-d(AGTTGAG(1)G(2)G(3)G(4)ACTTTCCCAGCC)-3'. We have observed that substitution of 8-oxo-dG at the G(1) site increases p50 binding affinity by approximately 2.5-fold compared to that of the unmodified DNA sequence, while substitution at G(3) reduces the binding affinity by approximately 4-fold. Substitution of 8-oxo-dG at the G(2) and G(4) sites had a minimal impact on p50 binding affinity. Both Escherichia coli fapy glycosylase (Fpg) and human 8-oxo-DNA glycosylase (hOGG1) recognized and cleaved 8-oxo-dG at all four sites within the promoter element. The addition of the p50 transcription factor shielded these lesions from cleavage by the glycosylase in a manner that correlated with the binding affinities of p50 for the different modified sites. These data imply that lesion formation in DNA response elements can modulate gene transcription during oxidative events and that protein binding to these modified sites may allow these lesions to persist on a time scale that impacts global cellular gene transcription.
Assuntos
Reparo do DNA , Desoxiguanosina/análogos & derivados , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , NF-kappa B/metabolismo , Transcrição Gênica/fisiologia , 8-Hidroxi-2'-Desoxiguanosina , Sequência de Bases , Sítios de Ligação , Sequência Consenso , DNA-Formamidopirimidina Glicosilase , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Escherichia coli/metabolismo , Humanos , N-Glicosil Hidrolases/antagonistas & inibidores , N-Glicosil Hidrolases/metabolismo , NF-kappa B/química , NF-kappa B/genética , Oxirredução , Regiões Promotoras Genéticas , Ligação Proteica , Subunidades Proteicas , Fatores de TempoRESUMO
The hexavalent oxidation state of chromium [Cr(VI)] is a well-established human carcinogen, although the mechanism of cancer induction is currently unknown. Intracellular reduction of Cr(VI) forms Cr(V), which is thought to play a fundamental role in the mechanism of DNA damage by this carcinogen. Two separate pathways of DNA damage, an oxidative pathway and a metal-binding pathway, have been proposed to account for the lesions observed in cell systems. We have used a model Cr(V) complex, N,N-ethylenebis(salicylidene-animato)oxochromium(V) [Cr(V)-Salen], to investigate the oxidative pathway of DNA damage and to elucidate the lesions generated from this oxidation process. Reaction of Cr(V)-Salen with synthetic oligonucleotides produced guanine-specific lesions that were not 8-oxo-2'-deoxyguanosine, based on the inability of iridium(IV) to further oxidize these sites. Oxidation products were identified using a 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-G) containing oligonucleotide to increase the yields of product for identification by electrospray ionization mass spectrometry. The guanine-based lesions observed by mass spectrometry corresponded to the lesions guanidinohydantoin and spiroiminodihydantoin. The effects of these Cr(V)-Salen-induced lesions on DNA replication fidelity was assayed using a polymerase-based misincorporation assay. These lesions produced G --> T transversion mutations and polymerase stops at levels greater than those observed for 8-oxo-G. These data suggest a model by which chromate can cause DNA damage leading to mutations and cancer.