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DNA Damage and Repair

IN VIVO DNA BASE DAMAGE IN HYDROGEN PEROXIDE- TREATED MAMMALIAN CELLS Govind Rao, Zeena Nackerdien, B. C. Chao, Ewa Gajewski and Miral Dizdaroglu University of Maryland Baltimore County, Baltimore, MD 21228, and National Institute of Standards and Technology, Gaithersburg, MD 20899, U.S.A.

ASSESSMENT OF DNA REPAIR (BY UDS) OF OXIDATIVE DNA DAMAGE IN TWO MURINE LEUKAEMIC CELL-LINES. Shirley-Ann M. Richardson, Bernie M.Hannigan, and P.Gerry McKenna. Biomedical Sciences Research Centre, University of Ulster at Coleraine, Co.Londonderry, Northern Ireland. BT52 1SA

Excess generation of free radicals in vivo may result in damage to biomolecules including DNA. Little is known about the chemical nature of free radical-induced DNA damage in vivo. We report on the first chemical characterization of in vivo damage to nuclear DNA in mammalian cells caused by exposure to H202 . Chromatin was isolated from H202-treated cells and analyzed by GC/MS with selected-ion monitoring. Following DNA base products were identified and quantitated: 5-methyl-5-hydroxyhydantoin, 5-hydroxyhydantoin, 5-hydroxymethyluracil, cytosine glycol, thymine glycol, 5,6-dihydroxy- cytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5- formamidopyrimidine and 8-hydroxyguanine. The yields of these products were dependent on H202- concentration. The methodology used permitted the analysis of these compounds directly in chromatin without the necessity of isolation of DNA from chromatin. Modified bases identified were typical hydroxyl radical-induced products of DNA bases, well known from in vitro studies. Results indicate involvement of hydroxyl radical in the mechanism of nuclear DNA damage in mammalian cells caused by H202 .

The capacity of a cell to repair oxidative damage is important since damage to DNA may lead to transformation. Unscheduled DNA synthe- sis(UDS) was used to assess excision repair ability in two mnrine cell-lines,707 Friend and 707BUF ce!is(707BUF is a thymidine kinase deficient (TK) derivative of 707). Oxidants were generated enzymatically(xanthine/xanthine oxidase,x/xod). Cell survival was assessed by cloning and endogenous antioxidant levels were measured. Cell survival was decreased in a dose dependent manner,particularly in 707BUF cells,following treatment. UDS showed that both cell types exhibited equal amounts of excision repair.Repair following higher levels of x/xod was greater in 707 cells.707 and 707- BUF cells had similar amounts of catalase but the 707BUF cells had 25% less superoxide dis- mutase(SOD) than 707 cells. It is proposed that excision repair occurs, the extent of which depends upon the cell's susceptibility to oxidants, endogenous antioxidant levels, and excision repair capability.

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5.31 HYDROXYL RADICAL GENERATION BY OLIGONUCLEO- TIDE DERIVATIVES OF ANTHRACYCLINE ANTIBIOTIC AND SYNTHETIC QUINONES Galina V. Rumyantseva, Sergei I. Dikalov, and Lev M. Weiner Institute of Chemical Kinetics and Combustion, Novosibirsk, 630090, USSR

To increase the specificity of the action of anticancer anthra- cycline drugs and their potential synthetic analogues, it is proposed to covalently bind them to oligonudeotides of different sequences. New compounds such as daunomycin (Din) or synthetic napthoquinone (NQ) covalently bound to heptadeoxynucleotides pCCAAACA (Dm-pN r) and deca- deoxythymidylate (pT10p-NQ) were prepared. These oligo- nudeotide derivatives can form specific complexes with complementary oligonucleotide sequences. It was found that these compounds can be reduced in a microsomal electron transfer chain as well as with purified NADPH-cytochrome P-450 reductase. Using the spin trap 5,5-dimethyl-l-pyrroline N-oxide, it has been shown that Dm-pN7 and pTmp-NQ are capable of generating hydroxyl radicals with or without complementary oligonucleotides. The mechanism of hydroxyl radical formation by these new compounds is described quite well by the previous scheme [G.V. Rumyantseva and L.M. Weiner, FEBS I.~tt. ~ 459-463 (1988)]. The stability of these compounds in redox reactions was studied. The oligonucleo- tide derivativres of anticancer and synthetic quinones are shown to have a high potential for site-specific scission of DNA of normal and tumor cells.

OXIDATION OF DNA BY PHOTOIONISATION, BY REACTION WITH SO,WAND WITH OH RADICALS. FREE RADICAL IN'I~RMEDIATES IN AQUEOUS SOLUTION.

D. Schulte-Frohiinde and K. Hildenbrand Max-Planck-Institut f/Jr Strahlenchemie

Stiftstr. 34, D4330 M/ilheim a.d. Ruhr, FRG.

With EPR we have identified neutral base radicals formed as intermediates by one-electron oxidation of double-stran- ded (ds)DNA from calf thymus at 20°C. The oxidation was achieved by UV light-induced photoionisation or by reaction with SOa ~- radical ions. It is suggested that upon photoionisation of DNA mainly the guanine radical cation, G ÷', is generated similar as in frozen solutions. 1 By rapid proton transfer from G +' to the hydrogen-bonded cytosine (k - 1012 s -1) G +" is converted into the deprotonated neutral radical G'(-H) which is detected by EPR. With SO4:-besides G'(-H) secondary radi- cals of the purine bases contribute to the spectra. By reaction of OH radicals with dsDNA in addition to G'(-H) the signals of the 6-OH adduct radical of thymine (6-hydroxy-5,6-dihydro-thymin-5-yl, 1 ) and of the allyl-type radical formed by H abstraction from the methyl group of thymine (5-methyleneuracil, 2 ) were produced. Time-resolved EPR measurements and pulse-conductivity data show that the thymine radicals 1 and 2 can be excluded as precursors of the fast, dominant component of strand breakage of beat-denatured, single-stranded (ss)DNA. One-electron oxidation with a Ru~+-complex leads to inactivation of the biological activity of plasmid DNA without strand break formation (with H. Steffen).

1)Sevilla,M.D., D'Arcy,J.B., Momhouse,K.M., and Engel- hardt,M.L., Photochem. Photobiol., 29, 37--42, (1979)

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