Antibodies to oxidative DNA damage: characterization of antibodies to 8-oxopurines.

The 8-oxo-7,8-dihydropurines (8-oxopurines) are important cellular premutagenic lesions produced in DNA by free radicals. Specific antibodies were prepared to detect these lesions. For antigens, 8-oxo-7,8-dihydroadenosine (8-oxoAdo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) were synthesized from the bromonucleosides, and the immunogens were produced by conjugating these to either bovine serum albumin or rabbit serum albumin by the periodate method. Polyclonal antibodies specific for the haptens were elicited from rabbits immunized with the BSA conjugates. The antibodies to 8-oxoAdo (anti-8-oxoAdo) and 8-oxoGuo (anti-8-oxoGuo) precipitated the homologous antigens in an Ouchterlony gel diffusion assay and no cross-reactivity was observed toward the normal nucleosides or to the heterologous 8-oxopurine. Specificity was also examined by hapten inhibition of antibody reactivity with the homologous conjugates using ELISA. For anti-8-oxoAdo, the IC50 for 8-oxoAdo was 8 mumol/L and 8-bromoadenosine, guanosine, and inosine did not inhibit, even at concentrations of 1.25 mmol/L. Similarly, the IC50 for anti-8-oxoGuo for 8-oxoGuo was 0.1 mumol/L. 8-Methoxyguanosine also inhibited the reaction but was about 500-fold less effective than the eliciting hapten. Other nucleosides tested did not inhibit at concentrations up to 100 mumol/L. Both antibodies could easily detect the corresponding damage in x-irradiated f1 DNA at a dose of 7.5 Gy and both antibodies recognized the corresponding lesion in duplex DNA; however, with anti-8-oxoGuo the signal was reduced about 50% compared to single-stranded DNA. In order to determine the exact amount of each lesion produced in irradiated DNA, and to standardize the ELISA signal, both products were measured after alkaline phosphatase digestion of x-irradiated calf thymus DNA using high-pressure liquid chromatography (HPLC) coupled to an electrochemical detector. Anti-8-oxoGuo could detect ten 8-oxoG residues and anti-8-oxoAdo could detect two 8-oxoA residues per 10,000 nucleotides. Thus, these antibodies should be useful for the detection and measurement of 8-oxopurines in cellular DNA.

Kinetics of radiation-induced strand break formation in single-stranded pyrimidine polynucleotides in the presence and absence of oxygen; a time-resolved light-scattering study.

Time-resolved reductions in the light-scattering intensity (LSI) of aqueous oxic and anoxic solutions of poly-C and poly-U at pH 7.8, following pulse-irradiation, have been studied as indices of strand break formation. With doses of 3-24 Gy per pulse, a number of kinetically distinct strand breakage components have been detected. A comparison of the LSI responses obtained from irradiations conducted under N2O with those conducted under air or O2 show no marked difference in the overall extent of LSI change. However, the immediate and fast (t 1/2 less than or equal to 50 microseconds) reduction in LSI, accounting for about 18-19% of the pyrimidine polynucleotide's total LSI response in oxic solution, is reduced in the absence of oxygen, to about 12% of the total LSI response found with poly-C and to about 9% for poly-U. For poly-C there is a five-fold enhancement in the rate of major strand breakage under anoxia [k1(N2O) = 7.9s-1] whereas for poly-U a more modest enhancement (about two-fold) is observed. These enhanced rates are mirrored by those for the losses of the principal optical anoxic absorptions (observed pulse radiolytically) that are assigned to the pyrimidine 6-yl base radicals. Such findings support a proposal that the rate-limiting step of major strand breakage for pyrimidine polynucleotides is a base radical mediated hydrogen atom abstraction reaction (Lemaire et al. 1987, Hildenbrand and Schulte-Frohlinde 1989). Irradiation of poly-C and poly-U in N2O/O2 (4:1, v/v) saturated solutions yields LSI changes much larger than those noted under N2O and air (or O2), which are in turn approximately double the responses observed under N2. This indicates that the major strand breaking species of water radiolysis is the OH-radical and that there is an oxygen enhancement of single strand breakage of about 1.9 for poly-C and 1.6 for poly-U.

The kinetics of radiation-induced strand breakage in polynucleotides in the presence of oxygen: a time-resolved light-scattering study.

The time-resolved light-scattering changes of aqueous, aerated solutions of poly-C, poly-U and poly-A at pH 7.8, following pulse irradiation, have been studied as indices of strand break formation. With doses of 4-24 Gy/pulse a number of kinetically distinct components have been detected. For the poly-pyrimidines an immediate and fast reduction (tau 1/2 less than or equal to 50 microseconds) in light-scattering intensity (LSI), accounting for approximately 20% of the total LSI change, is followed by a much slower loss (k1 approximately 1.6 s-1) which constitutes their major LSI change. For poly-A a similar fast component is observed, present to an extent equivalent to the one noted with poly-C; it constitutes, however, over 50% of the purine polynucleotide's total response, with the remainder of the change being a slower loss (tau 1/2 approximately 0.09 s). Optical pulse radiolysis studies of poly-C and poly-U, in support of the LSI investigations, show that transient absorbances in a region assigned to base peroxyl radicals decay in a complex fashion, with some at a rate equivalent to that for the slow (major) component of LSI loss. These observations support a proposal that the rate-limiting step of major strand breakage for these polynucleotides, in the presence of oxygen, is a base peroxyl radical-mediated abstraction of a H-atom from an adjacent sugar moiety (Bothe et al. 1986), with the resulting sugar peroxyl radicals then leading to strand break formation at a rate equivalent to that for loss of the initial, fast LSI components. These latter processes are attributed to strand breaks arising from the direct interaction of .OH with the polynucleotide sugar phosphate backbone.

Mechanism of radiation-induced strand break formation in DNA and polynucleotides.

The present state of our knowledge of the OH radical-induced strand break (sb) formation in presence and absence of oxygen in aqueous solution is reviewed for poly(U), poly(C), poly(A) and single- and double-stranded DNA as substrates. It was shown earlier that a single OH radical can induce a double-strand break in DNA. As a key step in the mechanism an interstrand radical transfer is postulated. The OH radical reaction is part of the indirect effect of gamma-irradiation. In addition recent results are presented concerning sb formation by the direct effect of high-energy irradiation using laser-induced photoionization for the formation of radical cations.

Template properties of ultraviolet-irradiated poly(dC) replicated by E. coli DNA polymerase I: indication for a role of apyrimidinic-sites in UV-induced mutagenesis.

Ultraviolet irradiation alters the template properties of poly(dC) when replicated by Escherichia coli DNA polymerase I. These effects are due to base modifications. Some of them are identified as apurinic/apyrimidinic sites (AP-sites) by their sensitivity to AP-endonuclease B purified from Micrococcus luteus, and their template properties. The rate of formation of AP-sites in poly(dC) is estimated at 3 X 10(-7) site per nucleotide per J.m-2. Exposure of supercoiled or relaxed pBR322 DNA to UV light results also in the formation of sites sensitive to AP-endonuclease B. In this case, the rate of formation of AP-sites is the same in relaxed or supercoiled DNA: 0.3 X 10(-7) site per nucleotide per J.m-2. The apyrimidinic sites are generated through the processing of an ultraviolet induced primary lesion. We suggest that this lesion is cytosine hydrate by its rate of decay and preferential formation in single stranded DNA. Our results suggest that AP-sites might be a minor pathway leading to UV-induced mutagenesis.