Chem -BtoL Interactions, 76 (1990) 111--128 Elsevier Sclentlhc Pubhshers Ireland Ltd

ALTERNATING CURRENT OF DNA DAMAGE

VOLTAMMETRIC

111

DETERMINATION

D KRZNARIC', B (~OSOVI(~", J STUBER b and R K ZAHN b~

•Center for Mamne Research Zagreb, Ruder Bo~kow~ Instztute, B~jen~ka 55, ~1000 Zagreb, {Yugoslav~ bInst~tute for Phys~olegwal Chemistry, University of Ma~nz (F R G ] and cAcademy o/ Science and L~terature, 6500 Ma~nz fFR G] and Laboratory for Mamne Molecular Bwlogy, Ma~nz and Yu 52120 Rown3 fYugoslavu~] (Received December 6th, 1989) (Revmlon received June 12th, 1990) (Accepted June 19th, 1990)

SUMMARY

The conditions for alternating current (a.c.)voltammetrlc D N A determinations have been investlgated with respect to its use wlth alkaline filterelution techniques at low D N A concentrations. In inorganic electrolyte solutions three current peaks can be distinguished: peak I around -1.1 V caused by the reorientation or desorption of D N A segments; peak II around -1.2 V caused by the native D N A (nDNA) form; peak III caused by denatured D N A (dDNA) at -1.4 V. Sonication of n D N A increases the peak current, however not with dDNA. Both d D N A and n D N A give linear peak current Increments with D N A increments, their regression lines cutting the concentration axis at the origin. In filterelution techniques organic bases are often used. Adding ethanolamine (EA) elution buffer decreases the peak amplitude of D N A . It turns out that an unknown substance, perhaps a protein or R N A , elutes from the filtersand glves rise to a current peak at about -1.3 V. This substance can interfere with the d D N A by competing for electrode surface area, since it diffuses much faster than the large molecules of the D N A Since however, d D N A has a higher affinityfor the electrode surface, after enough time, usually few minutes, the d D N A increasingly displaces the substance and occupies the surface. The same is valid for other organic molecules and thus also for EA. It is therefore remarkable that the unknown substance can be altered by ultrasonication,so that it will no longer Interfere with dDNA, in contrast to EA. EA, on the other hand, can be "titrated". W h e n E A is present at short accumulation times it prevents d D N A adsorption. By adding dDNA, the E A can be scavanged and further addition will adsorb and thus increase peak current in proportion to the concentration of the D N A preAbbrevlatlons a c, alternating current, dDNA, denatured DNA, EA, ethanolamme, EDTA, ethylenedlamme tetraacetlcacld,nDNA, natlve DNA, VA, voltammetry, voltammetrm 0009-2797/90/$03 50 © 1990 Elsevmr Sclentlhc Pubhshers Ireland Ltd Printed and Pubhshed m Ireland

112 sent. The conditions for voltammetric DNA determination have been investigated obeying the recognized Interactions. Avoiding organic bases and using Inorganic ones would simplify the determination procedure The reproducibility of the procedure in the range of 50--60 ng DNA/ml has been found to be __.6%.

Key words DNA damage determination -

DNA voltammetr,c determination -- DNA sonlcation -- alkaline filter elutlon of DNA -- EthanolamineDNA interaction

INTRODUCTION The actual environments of hv,ng organisms always contained, and by anthropogenIc influence additionally acquire, agents that bear considerable potential for DNA alteration [1,2]. By far the most frequent ones are single strand breaks [3]. One of the best methods for assessment of this kind of damage is by alkaline filter elutlon technique [4]. This technique requires the use of about 2 x l0 s cells/cm2 of filter surface. Deviations from these figures usually lead to bad results However, using these figures also implies that the DNA eluted from the filter comes off m concentrations changing over a wide range but always ,n very high dilution There are in the meantime quite adequate methods for DNA determination using fluorescent dyes [5]. In many cases it would be desirable to have more sensitive methods allowing for higher precision especially in the higher dilution range of the filter elutlon technique. Generally, electrochemical methods satisfy such requirements r a t h e r well. Investigations of DNA and other natural macromolecules by electrochemmal methods were extensively reviewed by PaleSek [6--9]. A.c. voltammetric measurements have been envisaged for possible use in filter elutlon techniques [10]. Among other questions the one of DNA measurement under the alkaline conditions prevailing in the elution system has been ventilated MATERIALS AND METHODS All chemicals were of the highest purity available. EthanolamIne came from Merck (Darmstadt, (F.R.G.)). All DNA used in the experiments was from Holthuma tubulosa males. It has been prepared by the method of Zahn et al [11]. It contained less than 1% of protein and RNA Measurements were carried out with a Polarecord E 506 (Metrohm Herlsau, Switzerland) in connection with a Metrohm Multi-Mode electrode (used as hanging mercury drop electrode}, an Ag/AgCl (3 mol/1) reference electrode

113 and a glassy carbon auxiliary electrode. The surface area of the hangingmercury drop electrode was 0.0041 cm 2 Throughout the experiments a Metrohm glass cell of 10 ml has been used. The solution was stirred with a rotating teflon coated magnetic bar Unless otherwise stated, the rotation speed was 3000 rev./min. After stirring, the solution was allowed to quiet down for 30 s prior to applying potenhal scan. Experiments were carried out at room temperature. The solutions were deaerated with nitrogen prior to measurements. Unless otherwide stated, all alternating current voltammetrm measurement were carried out at a frequency of 75 Hz and an amphtude of 10 inV. The phase angle was kept at 0 ° i.e. the in-phase component of the current was measured. The working solutions were prepared from 1 or 2 ml of the eluate sample, adding to it supporting electrolyte and making it up to 10 ml with quartzdistilled water. Two DNA stock solutions were prepared: (A) in "SSC" buffer: NaC1 0.15 mol/1, Sodium Citrate 0.015 mol/1, EDTA 0 02 tool/1 (pH 7); (B) EDTA 0 02 mol/ l, NaOH to bring it to pH 13. (C) (working solution) NaCI 0.3 tool/l, NaHCO 3 0.03 mol/1 (pH 8 9) (A) has been used to keep the DNA in its native state, while m (B) it was denatured. Final DNA solutions have been prepared by using 2 ml with the chosen amount of DNA. This is then brought to 10 ml with working solution, buffer C, if necessary along with the other desired components. THE ALKALINEELUTIONTECHNIQUE In this technique suspensions from cell cultures can be used as well as homogenates from organs. Essentially the technique of Kohn et al [4] has been followed with some modlhcatmns. In brief: The suspensmn finally is made up in buffer D (EDTA 10 mmol/1, NaHSO 3 5 mmol/1, KC1 1450 mmol/l, pH 7 4). This suspension at a density of about 1-2 mllhon cells is pumped at a speed of 0.2 ml/mln onto PVF filters (Millipore, Neu-Isenburg, F R.G.) m filter holders, always taking care of keeping the whole system free of air bubbles, followed by 5 ml of ice cooled buffer E (NaCl 140 mmol/1, KH2PO 4 1.5 mmol/1, KC1 2.7 mmol/1, Na2HPO 4 8.1 mmol/1, EDTA 0.53 mmol/1, pH 7 4) Subsequently, lyslng buffer F (Sodlum-Laurylsarcoslnate solution 2% (v/v), NaC1 2 molfl, EDTA 20 mmol/l, pH 10) is pumped at a speed of 0.2 ml/mm until a total of 4.5 ml have passed. This is then followed by 10 ml of a washing buffer G (EDTA 20 mmol/1, pH 10) at a speed of 0.2 ml/mm and finally by eluhon buffer H (EDTA 20 mmol/1, Ethanolamme (EA) to bring the pH to 12.3, which has to be prepared always fresh) at a speed 0.05 ml/mln until 18 ml have passed. It is collected in 3-ml fractions. The tubes are then flushed by soluhon H. These are the fractions, the DNA concentrations of which are characterizing the extent of DNA damage. In cases where the DNA concentratmn m all fractions is low, the

114 DNA has only few single strand brakes. The amount of DNA remaining on the filters is not considered m this investigation If, however, the DNA concentratmn m the first fractions Is h~gh and strongly decreasing towards the later fractmns, then damage must be h~gh. This behavmur may be modified ff DNA is cross-hnked to proteins that stink to the filter. The m e a s ur e m ent of the DNA concentration using fluorescent dyes poses some problems In those fractmns where the concentration m the eluted alkahne solutmn is very low -- and this coincides with those that had been marginally damaged only and whmh may be the most interesting ones, e.g. below 50 ng/ml ~t may become unmeasurable Even m the higher concentratmn ranges the quahty of the outcome depends among other points, on the temporal regime Th~s makes the measurements senmtlve to de~vatmns m timing of the different reaction steps. This point has been overcome by introducing automatm handling of the alkahne eluates Therefore the next effort is directed towards possible increase in DNA concentratmn de t e r m m at m ns using vol t am m et ry -

-

RESULTS AND DISCUSSION

The form of the potentzal-current relatwnsh~p of denatured D N A m 0 3 mol/l NaC~ 0 03 mol/l NaHCO s pH 8 9 Figure 1 gives typmal alternating current (a c.) voltammogram Two sharp peaks, one around - 1 . 4 V charactemstic of denatured DNA, peak III is m accordance with our previous paper [10] A smaller peak around - 1 . 1 V can be observed for both nDNA and dDNA and is ascribed either to a slow desporptlon of segments of DNA adsorbed mainly via sugar-phosphate backbone [7] or to the reormntatlon of the constituents of DNA [12]. This peak proved to be r a t h e r variable It has not been investigated any further A c voltammetmc compamson of native and denatured D N A zn ethanolamme containing solutwns Figure 2 demonstrates the dlfference in the potential vs current curves for native (A) and denatured DNA (B) dDNA was prepared by placing nDNA m a solution of the final composition: NaCl 0 3 mol/l, NaHCO 3 0.03 mol/1, EDTA 0.002 mol/1, ethanolamlne 0.002 mol/l (pH 12 3) for 10 mm This pH denatured nDNA. The pH has been lowered to pH 8 9. The same procedure was applied m the preparation of nDNA solution, except that nDNA was added at the end when pH was already 8 9 Native DNA gives t hr e e moderate peaks, denatured DNA two prominent ones. Theoretical speculation as well as expemmental observation (see later) implied the peak II of curve A being characteristic of nDNA, while ~ts peak III belongs to some denatured fractmn coming along with it Peak III ~s characteristm for dDNA Both peaks II and III from both curves can be used to derive a measure for DNA denaturation and DNA concentrations. Lmeamty

115

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Fig. 2 A c voltammetrzc curves for 350 ~g/l (A) native and (B) denatured DNA m 0 3 mol/l NaCI, 0.03 mol/l NaHC08, 0 002 mol/] EDTA and apprommately 0 002 mol/ (C2Hs)~NOH Accumulation time 2 min, accumulation potential - 0 8 V, pH 8 9

116 of p e a k I I I height and c o n c e n t r a t i o n of D N A is d e m o n s t r a t e d in p r e v , o u s p a p e r [10] By c o m p a r i n g the p e a k s I I I f r o m Figs. 1 and 2, it can be d e m v e d t h a t the p r e s e n c e of e t h a n o l a m l n e in Fig. 2 c o n m d e r a b l y lowers p e a k III. If increasing a m o u n t s of elutlon buffer ( E D T A 0.02 mol/l and E A to b r i n g p H to 12.3) w e r e added to the solutmn of c o n s t a n t c o n c e n t r a t m n of d e n a t u r e d DNA, p e a k III, c h a r a c t e r i s t i c for d D N A , d e c r e a s e s c o n s i d e r a b l y and b e c o m e s b r o a d e r . Without E A the s a m e e x p e r i m e n t does not show such an effect We thus conclude t h a t the p r e s e n c e of E A in a c o n c e n t r a t , o n d e p e n d e n t m a n n e r lowers the D N A d e p e n d e n t height of p e a k I I I A p e a k m a l k a h n e E A e l u a t e s a r o u n d - 1 3 V zs n o t d u e to D N A

Alkaline eluates with E A show often only a b r o a d p e a k a r o u n d - 1 . 3 (Fig 3, c u r v e 1) if to 1 ml of eluate, solution C is a d d e d up to 10 ml for m e a s u r e m e n t . In such a case the addition of small c o n c e n t r a t i o n s of internal s t a n d a r d d D N A i n c r e a s e s t h e p e a k at - 1 . 3 V, although less t h a n e x p e c t e d e v e n w h e n E A effects are t a k e n into consideration. This i n c r e a s e of - 1 . 3 V p e a k could easily be t h e r e a s o n for m l s j u d g e m e n t of this p e a k as b e i n g due to dDNA, especially since often no o t h e r D N A p e a k is o b s e r v a b l e u n d e r such condl-

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Fig 3 A c voltammetmc curves for alkahne eluate with (C2Hs)4NOH(1 ml of eluate and solution made up to 10 ml) m 0 3 tool/1 NaC1 and 0 03 mol/l NaHCO~, pH 8 9 Solution was tltrated with internal standard of (1) 0, (2) 212 and (3) 339 ~g/1 denatured DNA Accumulation potential - 1 0 V, accumulation time 1 mm

117 tlons. The dependence of such - 1 . 3 V peak height vs. amount of added dDNA is usually linear, however with a very small slope. Using this slope for extrapolation of the peak height without addition of internal standard would bring dDNA concentrations of 2--15 mg/1 instead of around 100 pg/1. Therefore this - 1 3 V peak is not attr:buted to dDNA The additmn of ,nternal standard rather superposes onto an unrelated preexisting peak. At the moment ,t is not clear why should the two peaks superpose at low additmns of DNA, since the standard peak III of dDNA is about - 1 4 V. Posruble explanation is that, at first, added DNA undertakes some sort of interaction, other than simple displacement, with the unknown substance adsorbed at the electrode resulting in an increase of peak at - 1.3 V. When all unknown substance is used up or simply starts to displace from the electrode, the standard peak III or dDNA starts to appear, With increasing accumulatmn time this - 1 . 3 V peak hrst increases and after 2--3 mm starts to decrease. This decrease may be attributed to competmon for adsorpt,on sites on the electrode surface. The peak probably comes from a substance passing through the filter m the course of the alkaline elutlon process along with the dDNA. The form and potentml of the peak point towards RNA [10]. Since dDNA adsorbs more strongly, probably existing however at a lower concentration and having a lower diffusion coefficient than the - 1 . 3 V peak substance, this will reach the electrode first. Yet with increasing accumulation times the DNA will displace it The same is observed ff dDNA concentration ~s increased. It is fortunate that the two peaks (at - 1.3 V and - 1.4 V) are suffic,ently separated once the peak III of dDNA appears. As can be seen from Fig 3, peak III height determination presents no difficulty. The interaction of the - 1 3 V substance w~th D N A

Proof that dDNA with increasing concentrations indeed displaces the - 1 3 V compound ~s m F~g. 3. A solutmn conta,nmg 1 ml of EA eluate made up to 10 ml with solution C gives one single prom,nent peak at - 1.3 V. With the addltmn of increasing amounts of dDNA th,s peak more and more decreases, while a new, a dDNA peak builds up around - 1 . 4 V, slgmfymg displacement of the umdentifmd - 1 . 3 V substance from the electrode surface. Substituting EA by NaOH m the alkahne elutlon buffer considerably increases the dDNA peak amplitude by factors of 5 - 1 0 w~thout ehmmatmg the interferences w:th adsorptmn competitors such as orgamc molecules in the filter eluates. Basically the alteratmn pattern of the dDNA peaks w,th increasing accumulation t,me ~s the same for the eluates with NaOH as w~th EA. In both cases the - 1 . 3 V and - 1 . 4 V peaks increase and at longer accumulation times decrease. More details are g~ven later. The onset of the - 1.4 V peak decrease occurs the sooner the more orgamc material other than DNA ,s involved including EA.

118

D~fferences among fractwns from the same filter elutwn Often, however, when filter eluates are measured, two peaks appear: the mentioned one around - 1 3 V and the standard peak III around - 1 . 4 V, charactemstm of dDNA. At high concentratmns of orgamc materml the peak III decrease can start already after short accumulatmn times, e.g. after 1 mm. This was often observed with fractmn number 1 of a filter eluate serms, whmh usually contains higher amounts of orgamc materml other than DNA. However, later fractmns of the same channel, containing less orgamc matereal, would give results with less interferences for the same accumulatmn t~me. It has been vemfied experimentally that by using short enough accumulatmn times (1 mm or less), expected values, as demved by other methods (fluorescent dyes}, may be approached more closely. The first fractions of different channels m filter elutmns qmte often ymld DNA values w~th high standard dewatmns. This Is paralleled by h~gh concentratmns of orgamc materml. Peak current dependence of d~fferent concentrations of added d D N A on accumulatwn tzme The interaction of the - 1.3 V substances and the peak III (around - 1.4 V) caused by dDNA has been discussed before. F~gure 4 gives another example for what can be detected by comparing different accumulation t~mes when h~gher dDNA concentrations are added to EA eluates. Since the - 1 . 3 V peak and peak III frequently supemmpose, addition of denatured DNA causing displacement of the - 1 3 V substance from the electrode, diminishes the - 1 . 3 V peak. This decrease however occurs faster, than the increase of the dDNA peak with the msmg DNA concentration, resulting m an overall decrease of the compound peak. Yet this hardly can explain the decrease of the compound peak to zero (Fig. 4, 4 ram). Here the effect of EA as the alkahmzatmn agent used m filter elutlon techmques comes into play. As demonstrated m Fig. 5 (curve 3) with short accumulatmn times, the DNA peak current m EA solutions, as occurring m filter elutlons, does not start to rose upon dDNA additmn unless more than 0.2 mg dDNA/1 are reached. Even then the slope is qmte shallow. Th~s is quite different with NaOH as alkah (Fig. 5, curve 2). Here the peak amphtude increase IS a steep function of the dDNA concentration m the solutmn, with the regressmn hnes intersecting at the origin. Practmally an ldentmal regressmn hne is obtained when no elutmn buffer was present m the solutmn (Fig. 5, curve 1) The effect of EA on the voltammetrm actlwty of dDNA may be explained by an anteractmn m solutmn resulting m a form that does not ymld peak III. Only after all free EA has been scavanged by dDNA, further dDNA addltmn gives peak III activity. The dependence of peak currents on accumulatmn times (F~g. 6) further illustrates that for short times the - 1 3 V substance within the first mm is increasing and then dropping to zero within the next three mm (curve 1),

119 7

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CONCENTRATION OF ADDED ONA ( p g / t ) F~g 4 Dependence of a c voltammetrm peak current (at - 1 3 V) on the concentratmn of added denatured DNA at varmus accumulation txmes DNA was added to a sample of 2 ml of eluate with (C2H6)4NOH (filled up to 10 ml of solution), 0 3 tool/1 NaC], 0 03 tool/1 NaHC08 (pH 8 9) Accumulatmn potentxal - 1 0 V

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