Biochemical Engineering Journal 22 (2004) 81–87

Quantitative assessment of DNA damage accompaniedwith no substantial loss in its molecular weight

during exposure to oxidative stress

SunYoung Kima, Takao Taguchia, Motomu Nishiokaa, Masahito Tayaa,b,∗a Division of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science,

Osaka University, Toyonaka, Osaka 560-8531, Japanb Research Center for Solar Energy Chemistry, Osaka University, Toyonaka, Osaka 560-8531, Japan

Received 7 July 2004; accepted 20 August 2004

Abstract

A pUC 19 plasmid DNA (2.7 kbp) was injured under the oxidative stress generated from TiO2 illuminated at incident light intensities of 9 and

1w toreaction,r1i ge in thea idatives njuredb talr©

K

1

gDtlbayssc

l

thelec-NAse, aseten-ing

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6 W/m2. The degree of DNA damage (D/D0) during the photoreaction was well expressed by a model equation,D/D0 = 1 − [1 − exp(−θ)

]P0,hereD0 andD are DNA densities at an original position estimated by agarose gel electrophoresis at an onset and a given time of pho

espectively, andθ is overall dose of oxidative stress to DNA. The parameter (P0) in this equation was determined to beP0 = 15.1 for the pUC9 plasmid irrespective of the examined light intensities. It was found that theP0 value depended on DNA molecular size, givingP0 = 10.3

n the case of a modified pUC 19 plasmid with an insertion of bacterial 16S rDNA (4.2 kbp). To estimate the degree of DNA damabsence of substantial loss in its molecular weight, theP value was defined as a remaining potential of DNA endurance against the oxtress, being formulated asP/P0 = exp(−θ). Transformation experiments were conducted by introducing the pUC 19 plasmid DNA iy the oxidative stress intoEscherichia coli, and it was found that the calculated value ofP/P0 could follow satisfactorily the experimenesults showing a gradual decrease in the transformation efficiency with increasingθ value in a region of 0 <θ ≤ 1.5.

2004 Elsevier B.V. All rights reserved.

eywords:TiO2 photoreaction; DNA damage; Reactive oxygen species; Oxidative stress; Transformation efficiency

. Introduction

It has been pointed out that the generation of reactive oxy-en species (ROS) and the subsequent oxidative damage ofNA can be critical elements in underlying events that lead

o mutation, cancer, and age-related degeneration of cellu-ar vitality [1–4]. It is known that DNA damage is inducedy superoxide anion (•O2

−) and nitric oxide (NO) as wells related oxidants, such as hydroxyl radical (•OH), perox-nitrite (ONOO−) and hydrogen peroxide (H2O2). Many re-earchers reported that DNA modification with mutagene-is was caused by these peroxy radicals[5,6] and derivedompounds including metal–oxygen complex[7], polyunsat-

∗ Corresponding author. Tel.: +81 6 6850 6251; fax: +81 6 6850 6254.E-mail address:taya@cheng.es.osaka-u.ac.jp (M. Taya).

urated fatty acid[8], metabolic by-product[9] and chemicaintermediate[10].

In the present study, we paid our main attention tosublethal DNA damage without substantial loss in its moular weight (i.e., no cleavage of covalent bonds in Dmolecules) during exposure to the ROS. For this purpophotoreaction system with TiO2 catalyst was chosen becauoperational conditions such as reaction time and light insity can be easily and strictly controlled by means of turnlight on/off. It is well known that various kinds of ROS (•O2

−,•HO, H2O2, etc.) are produced on TiO2 surfaces receivinnear ultraviolet rays (<410 nm), among which hydroxyl rical (•HO) is a dominant species responsible for the oxtive stress against living cells[11,12]. It was demonstratethat photoexcited TiO2 damaged plasmid DNA in vitro anchromosomal DNA in cultivated cells in vivo[13–15]. To

369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2004.08.005

82 S. Kim et al. / Biochemical Engineering Journal 22 (2004) 81–87

Nomenclature

D DNA density during photoreaction (mg/dm3)D0 DNA density at onset of photoreaction

(mg/dm3)E transformation efficiency by plasmid DNA

during photoreaction (–)E0 transformation efficiency by plasmid DNA at

onset of photoreaction (–)P remaining potential of DNA to endure oxida-

tive stress (–)P0 initial potential of DNA to endure oxidative

stress (–)S pixel numerals in DNA band at original posi-

tion on electrophoretic gel (–)

Greek lettersα strength of oxidative stress generated by pho-

toexcited TiO2 (min−1)θ overall dose of oxidative stress to DNA (–)θ0.98 θ value givingD/D0 = 0.98 (–)

our knowledge, however, there are few reports dealing withthe quantitative evaluation of DNA damage by ROS-derivedoxidative stress. In the present article, bacterial plasmids asmodel DNA molecules were subjected to the TiO2 photoreac-tion conducted under varied conditions. Based on model con-sideration, a parameter was proposed to quantify the degreeof DNA damage, focusing on a damaging stage where break-down of DNA molecules is substantially negligible. Further-more, the proposed parameter was endorsed in a biologicalaspect through the transformation ofEscherichia coliby thedamaged plasmid DNA.

2. Materials and methods

2.1. DNA molecules used

A plasmid pUC 19 (2.7 kbp), which harbores the genesencoding �-lactamase (for ampicillin resistance) and�-galactosidase, was used as model DNA. To prepare enlargedmolecules of DNA (4.2 kbp), a 16S rDNA sequence ofProv-idensiasp. WW2[16] (DDBJ accession no. AB161460) wasinserted into the pUC 19 plasmid at aHindIII site. Both theplasmids were subjected to experiments after being purifiedby using a QIAGEN Plasmid Midi Kit (QIAGEN Inc., USA),a

f e-n byi dms with

70% ethanol, dried under a reduced pressure, and then dis-solved in 10 mmol/dm3 Tris–HCl buffer (pH 8.0) containing0.1 mmol/dm3 ethylenediamine tetraacetic acid (EDTA). Theconcentration of linearized plasmid DNA was determinedby means of measuring optical density at 260 nm with aUV–visible spectrophotometer (Model UV-160, ShimadzuCorp., Japan).

2.2. DNA damaging experiments in TiO2 photoreactionand electrophoretic analyses

The experiments to examine DNA damage were con-ducted at 25◦C in a siliconized glass test tube (15 mmin diameter and 105 mm in height) filled with 10 cm3

of 10 mmol/dm3 phosphate buffer (pH 7.8) containing3.8 mg/dm3 linearized plasmid DNA, together with 0.1 g/dm3

TiO2 particles (Degussa P25, Japan Aerosil Co., Japan), un-less otherwise noted. The test tube was irradiated from a hor-izontal side by using a tubular type of 20 W black light fluo-rescent lamp with effective wavelengths of 300–420 nm (typeFL-20S BL-B, Matsushita Electric Industrial Co., Japan). In-cident light intensity,I, was adjusted to 9 or 16 W/m2, anaverage of values measured at several points on the tube sur-face with a quantum sensor (type J-221, UVP Inc., USA),by changing the distance between the lamp and tube. Atp ture(tar . Thep -l and0 ore-s eb -t andso edw )t suret red Im-a ityw d asf

D

w ec-t oseg

theo alka-l ture[ t thep bove,w -

ccordng to the supplier’s instruction.Each plasmid was linearized by digesting withBamHI

or 17 h at 37◦C and then treated with a mixture of phol/chloroform/isoamylalcohol (25:24:1, v/v/v), followed

sopropanol precipitation in the presence of 0.3 mol/3

odium acetate. The precipitated DNA was rinsed

rescribed exposure time, an aliquot of reaction mix300 mm3) was withdrawn from the test tube, and TiO2 par-icles were removed by centrifuging twice for 10 min at 4◦Cnd 15,000× g. Supernatant of the solution (200 mm3) wasecovered and condensed by isopropanol precipitationrecipitated DNA was dissolved in 10 mm3 of gel-loading so

ution (0.1% sodium dodecyl sulfate (SDS), 5% glycerol.005% bromophenol blue), and subjected to eletrophis on a 1.5% agarose gel in 40 mmol/dm3 Tris–acetatuffer (pH 8.0) containing 1 mmol/dm3 EDTA. The resulant gel was stained with ethidium bromide and the bf DNA molecules at an original position were visualizith a UV illuminator (Model LM-20E, UVP Inc., USA

o trace the change in DNA density with elapse expoime, as typically shown inFig. 1. The band images weigitalized using an image analyzing software (Scionge for Win, Ver.�4.02, Scion Co., USA), and DNA densas given by a predetermined calibration line expresse

ollows.

= 5.8 × 10−6 × S2 + 4.5 × 10−3 × S (1)

hereD andSare DNA density and pixel numerals, respively, in the DNA band at an original position on the agarel.

To check the looseness of DNA strands sufferingxidative stress, agarose gel electrophoresis under an

ine condition was also performed according to the litera17]. The samples of condensed DNA, which underwenhotoreaction and recovering treatment as mentioned aere introduced into 10 mm3 of gel-loading alkaline solu

S. Kim et al. / Biochemical Engineering Journal 22 (2004) 81–87 83

Fig. 1. Change in electrophoretic gel images of plasmid DNA with elapsedexposure time during TiO2 photoreaction. The pUC 19 plasmid was sub-jected to the photoreaction atI = 16 W/m2.

tion (50 mmol/dm3 NaOH, 1 mmol/dm3 EDTA, 0.1% SDS,5% glycerol and 0.005% bromophenol blue) and then ap-plied on a 1.5% agarose gel with 50 mmol/dm3 NaOH and1 mmol/dm3 EDTA, followed by eletrophoresis in alkalinesolution (50 mmol/dm3 NaOH and 1 mmol/dm3 EDTA). Af-ter the eletrophoresis, the gel was neutralized in 1 mol/dm3

Tris–HCl buffer (pH 7.6) containing 1.5 mol/dm3 NaCl andstained with ethidium bromide for visualization under UVillumination.

2.3. Estimation of intactness of damaged plasmid DNAin gene expression

A reaction mixture (800 mm3) from the TiO2 photoreac-tion conducted under a given condition was divided into twoaliquots. An aliquot (300 mm3) was subjected to the elec-trophoresis to determine the DNA density obtained from thetime-lapsed image on the agarose gel by the same method asmentioned above. The DNA solution recovered from the otheraliquot (500 mm3) was concentrated by isopropanol precipi-tation and dissolved again in 20 mm3 of deionized water. Apart of the DNA solution (10 mm3) was self-ligated by usinga Ligation-Convenience Kit (Nippon Gene Co., Japan) andthen applied for transformingEscherichia coliDH5�. Usingthe residual solution (10 mm3), DNA concentration in eachs eanso chst3

o 0 gp r cu-ba giono e of� d ontg l-� -

ducer, and then incubated for 40 h at 37◦C. Transformationefficiency,E, for each target gene was calculated from thenumber of developed colonies as follows:

E =

number of colonies on LB agar plate with ampicillin

or with X-gal

number of colonies on conventional LB agar plate(2)

3. Results and discussion

3.1. Time profiles of DNA damage by oxidative stressfrom photoexcited TiO2

The injuring of DNA molecules under the oxidative stresswas examined using the 2.7 kbp plasmid (pUC 19) in the TiO2photoreaction at different light intensities.Fig. 2 shows thetime profiles of relative DNA density at an original positionon the gel,D/D0, obtained from DNA samples in the pho-toreaction atI = 0, 9 or 16 W/m2. It was found that changein theD/D0 value was not appreciated in the darkness (I =0) for the examined exposure time untilt = 300 min. Underthe light condition (I = 9 or 16 W/m2), on the other hand, theD/D values decreased gradually with elapsed time, depictinga lankt eD yedi icatet lesi juryma

3s

e ofD ith

F imed smidw

ample used for the transformation was checked by mf fluorometric measurement with bis-benzimide (Hoe3258 dye, Sigma Co., USA)[18].

TransformedE. coli cells were cultured for 16 h at 37◦Cn agar plates containing Luria–Bertani (LB) medium (1olypeptone, 5 g yeast extract, 5 g NaCl and 15 g agar peic decimeter) with or without ampicillin (50 mg/dm3 whendded) to evaluate the degree of DNA damage in a ref ampicillin resistance gene. To investigate the damag-galactosidase gene, the cells were separately plate

he LB plates containing 5-bromo-4-chloro-3-indolyl-�-d-alactopyranoside (X-gal, 17.0 g/dm3) as well as isopropy-d(−)-thiogalactopyranoside (IPTG, 28.4 g/dm3) as an in

0profile of steeper drop at the higher light intensity. In a b

est conducted in the absence of TiO2 particles, a drop in th/D0 value was negligible under the conditions emplo

n the present work (data not shown). These results indhat the TiO2 photoreaction deteriorated the DNA molecun a light-dependent manner, suggesting that the DNA in

ay be attributable to oxidative radicals like•HO and•O2−

s reported in a literature[11,12].

.2. Model consideration of DNA damage by oxidativetress

In general, it is considered that the oxidative damagNA is accumulated in its molecules in association w

ig. 2. Changes inD/D0 values of plasmid DNA with elapsed exposure turing photoreaction under different light conditions. The pUC 19 plaas subjected to the photoreaction atI = 0, 9 or 16 W/m2.

84 S. Kim et al. / Biochemical Engineering Journal 22 (2004) 81–87

strength of oxidative stress and exposure period to the stress.In the present study, a variable,θ, is then employed for stan-dardizing the overall dose of oxidative stress loading on theDNA molecules. As conceptually illustrated inFig. 3A, θ canbe expressed as a function of exposure period,θ = αt, whereα means the stress strength which relates to the generationrate of oxidative radicals in the TiO2 photoreaction under agiven conditions.

It is expected that the degree of DNA injury will be inher-ently identical under an equivalent dose of oxidative stress interms of theθ value. In the present work, the DNA damageis formulated as a function ofθ, an original form of whichwas derived to describe the impact of radiation on biologicalvitality [19].

D/D0 = 1 − [1 − exp(−θ)

]P0 (3)

whereP0 corresponds to an extrapolated intercept ony-axisfor a linearized line approximated whenθ goes to infinity.

Based on the above-mentioned consideration, the valuesof D/D0 in the photoreaction atI = 9 and 16 W/m2 were re-plotted against theθ value as shown inFig. 4. This figureinclude the results obtained from the several experimentalruns (runs 1 and 2 atI = 9 W/m2, and runs 3 to 5 atI =16 W/m2). It was observed that the data closely overlappedwith each other, regardless of the light conditions. By match-i d tof ,= of

F s inT gte

Fig. 4. Change inD/D0 values of plasmid DNAs with varied molecular sizesexpressed in terms ofθ value during TiO2 photoreaction under different lightconditions. Two kinds of the plasmids (original pUC 19 plasmid and one withthe insertion of bacterial 16S rDNA) were subjected to the photoreaction atI = 9 or 16 W/m2.

15.1. It was thus confirmed that the degree of DNA damagecan be standardized in terms of theθ value even though DNAmolecules receive different strengths of oxidative stress.

3.3. Effect of DNA molecular size on its damage byoxidative stress

To examine the effect of DNA molecular weight on theoxidative injury, the TiO2 photoreaction was conducted usingthe pUC 19 plasmid with the insertion of a bacterial 16SrDNA sequence.Fig. 4 also shows the plots of theD/D0value against theθ value in the case of the modified plasmid(see runs 6 and 7 atI = 16 W/m2). The profiles ofD/D0 valueswere also traced by Eq.(3)exhibiting the fair agreement withthe data (r = 0.996). TheP0 value for the modified plasmidwas given asP0 = 10.3, the value of which was smaller thanthat for the original pUC 19 plasmid (P0 = 15.1). This resultimplied that theP0 value can be a parameter for the oxidativedamage of DNA depending on its molecular size.

This study was intended to quantify the extent of DNAdamage caused by the oxidative stress employing the modelparameter. The oxidative radicals produced from photoex-cited TiO2 can attack random sites of DNA chain to loosenor partially cleave the DNA strand. Considering the resultsshown inFig. 4, as modeled inFig. 3B, it is postulated that theP ida-t xistsa NA( byd chain

ng Eq.(3) to these data, the solid line could be depicteollow the profiles ofD/D0 values (correlation coefficientr

0.991), andP0 in Eq. (3) was determined to be a value

ig. 3. Conceptual drawing of DNA injury caused by oxidative stresiO2 photoreaction. Model consideration: (A) a viable,θ, for standardizin

he impact of oxidative stress on DNA damage, and (B) a parameter,P0, forvaluating the potential of DNA to endure oxidative stress.

D in-j small

0 value reflects the potential of DNA to endure the oxive stress when a certain amount of oxidative radicals eround the molecules. It is most likely that a long-chain Dhigh molecular weight) has a possibility to be attackedose of the oxidative stress at more sites than a short-NA, in other words, the long-chain DNA is liable to be

ured by the stress and consequently shows a relatively

S. Kim et al. / Biochemical Engineering Journal 22 (2004) 81–87 85

value ofP0. In the present work, theP0 value is proposed as aparameter for evaluating the degree of DNA damage causedby the oxidative stress. In this context, theP value is definedas a remaining potential of DNA endurance against the ox-idative stress and correlated with theθ value as given by thefollowing equation.

P/P0 = exp(−θ) (4)

3.4. Transformation efficiency with plasmid DNAdamaged by oxidative stress

As seen inFig. 4, the values ofD/D0 hardly changed atthe early stage of the photoreaction, meaning that the sub-stantial loss in molecular weight of plasmid DNA did notoccur during this period. However, it is most likely that theoxidative damage is accumulated in the DNA molecules evenat the stage without breakdown or fragmentation of the DNAchain. Our attention was then focused on the DNA injury bythe oxidative stress in a region up toD/D0 = 0.98 given atθ= θ0.98. In the case of pUC 19 plasmid shown inFig. 4, theθ0.98 value was calculated asθ0.98 = 1.5 by Eq.(3) with P0 =15.1.

To examine the progress of DNA injury (e.g., nick for-mation on the DNA chain) in the region of 0 <θ ≤ 1.5, theagarose gel electrophoresis was carried out under an alkalinec d atθ

n ntsb esisa t ob-sas re-v ti plea

F atives lec-t ectedt0 ductedu

To evaluate the intactness in gene expression of the in-jured DNA molecules in the region of 0 <θ ≤ 1.5 as men-tioned above, the pUC 19 plasmid recovered from the pho-toreaction (run 5 shown inFig. 4) was introduced intoE. colihost cells and the phenotypes of transformants were investi-gated in terms of the gene expression of�-lactamase and�-galactosidase.Fig. 6shows the relationship between the val-ues ofθ andE/E0 which is a ratio of transformation efficiencyat a givenθ value to that atθ = 0, exhibiting the same tendencywith respect to both the genes of interest. It was confirmed thatthe transformation efficiency did not changed when the plas-mid was treated with TiO2 in the absence of light under theexamined conditions (data not shown). These results stronglysuggest that the DNA injury by the oxidative stress progresseseven if the substantial loss in DNA molecular weight is notassociated with the oxidative reaction. It is well documentedthat guanine (G) is readily oxidized because of its low re-dox potential[21], and specifically that a 5′-site of GGGand GG sequences in double-stranded DNA is the most sen-sitive against oxidization to yield 8-hydroxydeoxyguanosine(8-oxodG)[22–25]. Several researchers reported that the con-secutive G residues acted as trapping sites in long-range ox-idative damage of DNA through one-electron oxidations ofdouble-stranded DNA[26,27]. Moreover, it was reported that8-oxodG formation can cause a transversion of guanine tot on[ -ln witht thed tion( nee

eterw agea ress.T eninga antsa DNA

F ass ni df

ondition using the samples of pUC 19 plasmid obtaine= 0 and 0.54. According to the report of Okada et al.[20],icked DNA is denatured to cut into single-chain fragmey alkali treatment and that DNA bands on electrophorre broadened. As expected, notable difference was noerved between the band patterns of DNA samples atθ = 0nd 0.54 on the neutral agarose gel (Fig. 5A). Meanwhile, ashown inFig. 5B, the alkaline agarose gel electrophoresisealed that the bands of DNA sample atθ = 0.54 broaden ounto a blurred pattern, as compared with that of DNA samt θ = 0.

ig. 5. Electrophoretic gel images of plasmid DNA exposed to oxidtress in TiO2 photoreaction under: (A) neutral condition (conventional erophoresis) and (B) alkaline condition. The pUC 19 plasmid was subjo the photoreaction atI = 16 W/m2, and the DNA samples recovered atθ =and 0.54 were loaded on the agarose gel for the electrophoresis connder each condition (see Section2).

imine via DNA misreplication with an outcome of mutati28,29]. The solid line drawn inFig. 6 indicates the calcuated value ofP/P0 using Eq.(4) with P0 = 15.1. It is worthothing that the calculated result is in chose coincidence

heE/E0 value (r = 0.989). It was thus demonstrated thategree of DNA injury estimated by the model consideraP/P0) could account for the deterioration of DNA in gexpression caused by the oxidative stress.

In the present study, we proposed the model paramhich permitted the quantitative assessment of DNA damccumulation in its molecules exposed to the oxidative sthe parameter presented can be a useful tool for the scrend valuation of DNA sheltering agents such as antioxidnd the understanding of their mechanisms protecting

ig. 6. Relationship betweenE/E0 andθ values. The pUC 19 plasmid wubjected to the photoreaction atI = 16 W/m2 (corresponding to run 5 show

n Fig. 4and the DNA samples recovered at the indicatedθ values were useor transformingE. coli (see Section2).

86 S. Kim et al. / Biochemical Engineering Journal 22 (2004) 81–87

molecules from oxidative damage. From this aspect, furtherwork will be undertaken to extend the results found in thepresent study.

4. Concluding remarks

The plasmid DNA molecules were damaged by the oxida-tive stress in the TiO2 photoreaction (I = 9 or 16 W/m2), andthe followings were concluded.

(1) The degree of DNA damage,D/D0, was satisfacto-rily expressed by the model equation,D/D0 = 1 −[1 − exp(−θ)

]P0, and the parameter was determined tobeP0 = 15.1 for the pUC 19 plasmid (2.7 kbp) regardlessof the examined light conditions.

(2) TheP0 value was dependent on the DNA molecular sizeand the value ofP0 = 10.3 was obtained for the modifiedpUC 19 plasmid with the insertion of bacterial 16S rDNA(4.2 kbp).

(3) The value ofP/P0, formulated as a function ofθ value,was proposed to quantify the extent of DNA damage atthe early stage of photoreaction where the breakdown ofDNA molecules was not of significance.

(4) The calculated value ofP/P0 was in fair agreement withthe experimental findings that the transformation effi-

as

s.

A

em-p E)p sents tificR theM ch-n

R

mul-. 371

.M.e intined

.S.A.

ionle of(1996)

an,sion,

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[ Hi-lysed–90.

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[ in

[ , Bi-DC,

[ frag-5–

[ in-tects04)

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[ the98)

[ leav-tionB-

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ciency with the damaged DNA (pUC 19 plasmid) wgradually deteriorated with an increase in theθ valueranging from 0 to 1.5 under the examined condition

cknowledgments

One of the authors (S.Y.K.) expresses his thanks forloying as a fellow in the Center of Excellence (21 COrogram “Creation of Integrated EcoChemistry”. The pretudy was supported in part by a Grant-in-Aid for Scienesearch on Priority Areas (417) (no. 15033241) froministry of Education, Culture, Sports, Science and Teology, Japan.

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