Phorochumisrr?. und Phorobiology Vol. 33. pp. 251 to 252 0 Pergamon Press Ltd 1981. Printed in Great Britain

003 1-865518 l/020 1-025 I Io2.00/0

RESEARCH NOTE

DNA-DENATURING EFFECT OF IRRADIATED HYDROGEN PEROXIDE

DANIEL ROTH Goodwin Institute for Cancer Research, 1850 N.W. 69th Avenue, Plantation,

FL 33313, USA

(Received 12 June 1980; accepted 19 August 1980)

Abstract-By an acridine dye method for analyzing DNA conformation, it was shown that hydrogen peroxide, in conjunction with irradiation at wavelengths longer than 290 nm, exerted a denaturing effect on linear duplex DNA. The effect was dose-related to a disappearing limit of 10 pM hydrogen peroxide.

INTRODUCTION

Hoffmann and Meneghini (1979) reported that the introduction of hydrogen peroxide (H202) to culture media at concentrations above 10pM reduced the survival rate of Xeroderma pigmentosum-derived fibro- blasts to a greater degree than that of control human cells. DNA extracted from both cell strains after treat- ment showed no significant differences in the inci- dence or repair rates of single-strand breaks (ssb), thereby pointing away from ssb as a cause of the dissimilar cell sensitivities to H 2 0 2 . These authors found that the production of ssb required cell metab- olism since they were not observed in purified DNA that had been treated directly with H 2 0 2 ; it was sug- gested that the active species may be oxygen radicals such as OH' o r 0; formed from H202 metabolically. The possible existence of a relationship between the sensitivity of Xeroderma cells to H20, and their well known sensitivity to UV radiation was not dis- cussed. Any correlation in this regard remains to be determined.

Regional denaturation of DNA could provide that link, for it occurs around pyrimidine photodimers, on the one hand, and may well result from certain forms of base damage due to H 2 0 2 on the other. Various lesions apart from ssb have been ascribed to HzOz, principally through its active oxygen radicals. These include ring destruction, deamination, and dehydrox- ylation of bases (Butler and Conway, 1953), liberation of adenine (Yamafuji and Uchida, 1966), breakage of double bonds in the bases and base liberation by oxi- dation of the C-1 carbon atom in deoxyribose (Rhaese and Freese, 1968; Rhaese, 1968), scission of double strands, base destruction, and cross-linking (Massie ef a/., 1972). However, their ability to denature DNA has not been established.

By employing methodology not previously used in determining the effects of H 2 0 z on DNA conforma- tion, the study being reported here attempted to gen- erate new and confirming data relating to the induc-

tion of denaturation. The procedural approach was based on the differential thermal stabilities of DNA- acriflavine bonds at single-stranded and double- stranded sites (Roth, 1973; Roth and London, 1977). Thus, altered thermal dissociation kinetics of the bonds reflect the relative extent of single-strandedness in a sample of DNA. This is expressed by changes in the fluorescence yield of dye upon being released from quenching sites, an analytical technique which was found to be more sensitive than the DNA melting profile or viscometry (Ostwald method).

Reaction mixtures contained purified linear duplex DNA- and H202. Active photoproducts of H202, presumably oxygen radicals, were generated by irradiation at wavelengths longer than 290 nm as a procedural alternative to their natural metabolic pro- duction (Butler and Conway, 1953).

MATERIALS AND METHODS

Aliquots of 1.5 pg of calf thymus DNA (Sigma Chemical Co., type I) were admixed with H202 (U.S.P.) at concen- trations ranging from M to M , in a total volume of 1 mf of 1.5 mM sodium chloride solution containing 0.2 mM sodium phosphate buffer, pH 6.8. They were then irradiated by a IOOW mercury lamp through a Kimax glass filter which cut off radiations shorter than 290nm. The incident fluence'was lo5 J/m2 in the spectral region between 290 and 334 nm, as measured by a Yellow Springs Instrument Co. radiometer, Model 65. A DNA-irradiation control and a DNA-H,02 dark control were included.

Each sample then received 0.16 nmol of acriflavine hy- drochloride (Matheson, Coleman and Bell) in 2 m/ of the buffered saline solution. These mixtures were allowed to interact in the dark with gentle shaking for 15min and were then transferred to hard borosilicate cuvets. in which their fluorescence intensity was immediately measured by a Photovolt fluorometer against a free dye standard and a solvent blank. A 438nm interference filter was in place across the exciting beam and a Corning glass filter, C.S. No. 4-105 across the emitted beam. The cuvets were then heated in a heating block at an increment rate of 2°C per min. and fluorescence intensity was measured at fixed tem- perature intervals. A limit was set at 65°C because dye was released rapidly from all the samples of DNA at higher

25 1

252 DANIEL ROTH

P

o+. 0 25 35 4 5 55 0 5

Temperature Figure I . Thermal dissociation kinetics of DNA-acrifla- vine bonds in terms of dye fluorescence intensity. Calf thymus DNA in 1.5 pg aliquots was irradiated by mercury arc at wavelengths longer than 290nm in the presence of HzOz at lo--’ M (0). M (A). and lo-’ M (0) concen-

trations. DNA-irradiation control (0).

temperatures as thermal denaturation of the DNA devel- oped from the heating process itself.

RESULTS A N D DlSCUSSlOli

In the absence of irradiation, H,O, was inactive. Figure 1 shows a dose-related incremental effect of HzOz in combination with constant irradiation, on the thermal dissociation kinetics of DNA-acriflavine bonds, to a disappearing limit of about 10pM H,02. The DNA-irradiation control incorporates a small DNA damaging effect from the radiation itself.

According to the rationale of the experimental tech- nique employed in this study, the data are taken to indicate that the extent of single-strandedness in the DNA samples increased with increasing H 2 0 2 con- centration. This interpretation must be modified to allow for an alternate concept in which the DNA structure may simply have been destabilized without undergoing complete denaturation. In this view the subsequent application of heat in the analytical pro- cedure would then induce strand separation at lower temperatures than normally, whereupon the specific dye response would ensue. Perhaps the two events occur in combination. interstrand bonds at the mar- gins of H,O,-denatured regions becoming weakened in consequence of their proximity to those regions and then rupturing at relatively low temperatures, thereby enhancing, to an unknown extent, the ‘unzip- pering’ action of the original experimental treatment.

Regional denaturation of DNA could comprise the basis of a common factor shared by UV- and H,O,-induced damage; the resulting distortion of DNA, such as loop formation, could provide recog- nition sites for an essential early function of various repair systems, including those involved in the repair of UV- and H,O,-induced damage. Inasmuch as ssb related to H 2 0 2 treatment seem to occur simply as a result of, and at a much lower rate than base damage (Rhaese and Freese, 1968; Massie et al., 1972), their efficient repair by Xeroderma cells, as ob- served by Hoffmann and Meneghini (1979), might not be paralleled by similarly efficient repair of denatured regions. Thus, Xeroderma cells, if in fact defective in this component of repair, would exhibit sensitivity to both UV radiation and H 2 0 2 .

REFERENCES

Butler. J. A. V. and B. E. Conway (1953) Proc. R . Soc. 141. B series. 562-580. Hoffmann, M. E. and R. Meneghini (1979) Phorochem. Phorobiol. 30, 151-155. Massie. H. R.. H. V. Samis and M. B. Baird Rhaese. H. J. and E. Freese (1968) Biochi~ii. Biopliys. Acrtr 155. 476490. Rhaese. H. J. (1968) Biochirii. B ioph~s . Acrcr 166. 31 1-326. Roth. D. (1973) Pliorochori. PhotohioL 18. 437-439. Roth. D. and M. London (1977) J. Iiirest. Deirtiaro/. 69, 368-372. Yamafuji. K . and Y. Uchida (1966) Ntrrirrr 209. 301-302.

(1972) Biochirn. Biopliys. Act({ 272. 539-548.