RESUMEN
Exposure to aristolochic acid (AA), a component of Aristolochia plants used in herbal remedies, is associated with chronic kidney disease and urothelial carcinomas of the upper urinary tract. Following metabolic activation, AA reacts with dA and dG residues in DNA to form aristolactam (AL)-DNA adducts. These mutagenic lesions generate a unique TP53 mutation spectrum, dominated by A:T to T:A transversions with mutations at dA residues located almost exclusively on the non-transcribed strand. We determined the level of AL-dA adducts in human fibroblasts treated with AA to determine if this marked strand bias could be accounted for by selective resistance to global-genome nucleotide excision repair (GG-NER). AL-dA adduct levels were elevated in cells deficient in GG-NER and transcription-coupled NER, but not in XPC cell lines lacking GG-NER only. In vitro, plasmids containing a single AL-dA adduct were resistant to the early recognition and incision steps of NER. Additionally, the NER damage sensor, XPC-RAD23B, failed to specifically bind to AL-DNA adducts. However, placing AL-dA in mismatched sequences promotes XPC-RAD23B binding and renders this adduct susceptible to NER, suggesting that specific structural features of this adduct prevent processing by NER. We conclude that AL-dA adducts are not recognized by GG-NER, explaining their high mutagenicity and persistence in target tissues.
Asunto(s)
Adenina/análogos & derivados , Ácidos Aristolóquicos/toxicidad , Aductos de ADN/metabolismo , Reparación del ADN , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Mutagénesis , Mutágenos/toxicidad , Adenina/química , Adenina/metabolismo , Ácidos Aristolóquicos/química , Línea Celular , Aductos de ADN/química , Proteínas de Unión al ADN/metabolismo , Desoxiadenosinas , Genoma Humano , Compuestos Heterocíclicos de 4 o más Anillos/química , Humanos , Mutágenos/químicaRESUMEN
Aristolochic acids (AAs), major components of plant extracts from Aristolochia species, form (after metabolic activation) pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present here a fast, nonisotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition and analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.
Asunto(s)
Aristolochia/química , Ácidos Aristolóquicos , Aductos de ADN/análisis , ADN , Extractos Vegetales , Animales , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/farmacología , Ácidos Aristolóquicos/toxicidad , Cromatografía Líquida de Alta Presión , ADN/síntesis química , ADN/química , Aductos de ADN/química , Aductos de ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Fluorescencia , Células LLC-PK1 , Mutágenos/química , Mutágenos/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , PorcinosRESUMEN
Human 8-oxoguanine-DNA glycosylase (OGG1) is the main human base excision protein that removes a mutagenic lesion 8-oxoguanine (8-oxoG) from DNA. Since OGG1 has DNA glycosylase and weak abasic site (AP) lyase activities and is characterized by slow product release, turnover of the enzyme acting alone is low. Recently it was shown that human AP endonuclease (APE1) enhances the activity of OGG1. This enhancement was proposed to be passive, resulting from APE1 binding to or cleavage of AP sites after OGG1 dissociation. Here we present evidence that APE1 could actively displace OGG1 from its product, directly increasing the turnover of OGG1. We have observed that APE1 forms an electrophoretically detectable complex with OGG1 cross-linked to DNA by sodium borohydride. Using oligonucleotide substrates with a single 8-oxoG residue located in their 5'-terminal, central or 3'-terminal part, we have demonstrated that OGG1 activity does not increase only for the first of these three substrates, indicating that APE1 interacts with the DNA stretch 5' to the bound OGG1 molecule. In kinetic experiments, APE1 enhanced the product release constant but not the rate constant of base excision by OGG1. Moreover, OGG1 bound to a tetrahydrofuran analog of an abasic site stimulated the activity of APE1 on this substrate. Using a concatemeric DNA substrate, we have shown that APE1 likely displaces OGG1 in a processive mode, with OGG1 remaining on DNA but sliding away in search for a new lesion. Altogether, our data support a model in which APE1 specifically recognizes an OGG1/DNA complex, distorts a stretch of DNA 5' to the OGG1 molecule, and actively displaces the glycosylase from the lesion.