phosphorus-radioisotopes and 8-hydroxyguanine

As a new approach to DNA adductomics, we directly reacted intact, double-stranded (ds)-DNA under warm conditions with an alkylating mass tag followed by analysis by liquid chromatography/mass spectrometry. This method is based on the tendency of adducted nucleobases to locally disrupt the DNA structure (forming a "DNA bubble") potentially increasing exposure of their nucleophilic (including active hydrogen) sites for preferential alkylation. Also encouraging this strategy is that the scope of nucleotide excision repair is very broad, and this system primarily recognizes DNA bubbles.. CAX-Prelabeling is an emerging new technique for DNA adductomics, providing polar DNA adductomics in a practical way for the first time. Further study of the method is encouraged to better characterize and extend its performance, especially in scope and sensitivity.

Topics: Animals; Benzo(a)pyrene; Benzyl Compounds; Cations; Cattle; Chromatography, High Pressure Liquid; DNA Adducts; Ethylamines; Guanine; Humans; Nucleotides; Phosphorus Radioisotopes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Uracil

A major product of oxidative DNA damage is 8-oxoguanine. In humans, 8-oxoguanine DNA glycosylase (hOGG1) facilitates removal of these lesions, producing an abasic (AP) site in the DNA that is subsequently incised by AP-endonuclease 1 (APE1). APE1 stimulates turnover of several glycosylases by accelerating rate-limiting product release. However, there have been conflicting accounts of whether hOGG1 follows a similar mechanism. In pre-steady-state kinetic measurements, we found that addition of APE1 had no effect on the rapid burst phase of 8-oxoguanine excision by hOGG1 but accelerated steady-state turnover (k

Topics: Adenosine Triphosphate; Cross-Linking Reagents; DNA; DNA Damage; DNA Glycosylases; DNA-(Apurinic or Apyrimidinic Site) Lyase; Gene Expression; Glutaral; Guanine; Phosphorus Radioisotopes; Protein Binding; Protein Transport; Recombinant Proteins; Staining and Labeling

Benz[a]anthracene (BA) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) that are ubiquitous environmental pollutants. PAH carcinogenesis is explained by DNA adduct formation by PAH diol epoxide and oxidative DNA damage by PAH o-quinone. Benz[a]anthracene-trans-3,4-dihydrodiol (BA-3,4-dihydrodiol) is a minor metabolite but shows higher mutagenicity and tumorigenicity than parent BA. We confirmed that a BA o-quinone type metabolite, benz[a]anthracene-3,4-dione (BA-3,4-dione), induced oxidative DNA damage in the presence of cytochrome P450 reductase. Interestingly, we found that BA-3,4-dihydrodiol nonenzymatically caused Cu(II)-mediated DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine formation and the addition of NADH enhanced DNA damage. BA-3,4-dihydrodiol induced a double-base lesion of C and G at the 5'-ACG-3' sequence complementary to codon 273 of the human p53 tumor suppressor gene, which is known as a hotspot. The DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H(2)O(2) and Cu(I). Time-of-flight mass spectroscopic study suggested that BA-3,4-dihydrodiol undergoes Cu(II)-mediated autoxidation leading to the formation of its hydroxylated form of BA-3,4-dihydrodiol, capable of causing oxidative DNA damage. It is noteworthy that BA-3,4-dihydrodiol can nonenzymatically induce DNA damage more efficiently than BA-3,4-dione with metabolic activation. In conclusion, oxidative DNA damage induced by BA-3,4-dihydrodiol not only via quinone-type redox cycle but also via a new type of redox cycle participates in the expression of carcinogenicity of BA and BA-3,4-dihydrodiol.

Topics: Animals; Benz(a)Anthracenes; Catalase; Copper; Deoxyadenosines; DNA Damage; DNA Fragmentation; Dose-Response Relationship, Drug; Genes, p16; Genes, ras; Guanine; Humans; NAD; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Oxidative Stress; Phenanthrolines; Phosphorus Radioisotopes; Polycyclic Aromatic Hydrocarbons; Quinones; Tumor Suppressor Protein p53

DNA damage caused indirectly via reactive oxygen species generated during reductive activation of mitomycin C was evaluated. This oxidative DNA damage was measured by determining the formation of 8-hydroxyguanine in DNA exposed to chemically or enzymatically activated mitomycin C. The level of 8-hydroxyguanine was measured indirectly by determining formamidopyrimidine-DNA glycosylase-sensitive sites induced in plasmid DNA exposed to mitomycin C and directly by a 32P-postlabeling assay for the modified base. Activation of mitomycin C by sodium borohydride in air, by H2/Pt, or xanthine oxidase in N2 caused increases in the level of 8-hydroxyguanine. The extent of the increase varied according to the incubation conditions with the greatest increase being observed in DNA exposed to mitomycin C activated under hypoxic conditions. These results support a possible indirect mechanism for DNA damage caused by mitomycin C that is mediated by reactive oxygen species.

Topics: Anaerobiosis; Borohydrides; DNA; DNA Damage; DNA-Formamidopyrimidine Glycosylase; Guanine; Mitomycin; N-Glycosyl Hydrolases; Phosphorus Radioisotopes; Plasmids; Substrate Specificity; Xanthine Oxidase

A method for the sensitive detection of 8-hydroxyguanine residues in small amounts of DNA (0.2-2 micrograms) was developed. It comprises (i) the enzymatic hydrolysis of DNA to 2'-deoxyribonucleotide 3'-monophosphates, (ii) degradation of the bulk amount of normal purine and pyrimidine deoxyribonucleotides in the DNA digest by treatment with trifluoroacetic acid and hydrazine, respectively, under conditions retaining the structure of d(8-OH-G)p necessary for 5' phosphorylation by T4 polynucleotide kinase (PNK), (iii) 5' phosphorylation of d(8-OH-G)p by T4 PNK-catalyzed transfer of 32P from [gamma-32P]ATP, and (iv) 2D thin-layer chromatography on polyethyleneimine-cellulose sheets to purify and resolve 32P-postlabeled d(8-OH-G)p. Model experiments with mixtures composed of synthesized d(8-OH-G)p and DNA hydrolysate indicate that it is possible to detect one 8-hydroxyguanine residue out of 2 x 10(6) normal bases starting with 1 microgram DNA. The methodology, which allows for a further decrease of this detection limit, might be very useful for the sensitive detection of DNA damage induced by activated oxygen species in small amounts of DNA. We demonstrate the formation of 8-OH-G in DNA in vitro by low doses of 60Co gamma-rays.

Topics: Adenosine Triphosphate; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; DNA; Guanine; Phosphorus Radioisotopes