A C T A U N I V E R S I T A T I S L O D Z I E N S I S - FOLIA BIOCHIMICA ET BIOPHYSICA 7, 1990
Ryaaard Wiaderkiewiea, Zofia Walter
PULS E-POLAROGRAP HIC CH ARACTER ISTIC OF DNA MODI FIED BY ORG ANOPHOSPHO RUS INSECTICIDES IN VITR O
Pulse-polarographic investigations of DNA isolated from call thymus and'modified by organophosphorus insecticides (oi) were car-ried out. Following standards of 01 were used: malathion (0,0-dime- thyl dicarboxyethyl dithiophosphate), 'DDVP (0,0-dimethyl-0-dichloro- vinyl phosphate), methylparathion (0,0-dimethyl-0-4-nitrophenyl tio- nophosphate) and methylbromphenvinphos (0t0-diœethyl-0-1-2,4-dichlo- rophenyl-2-bromvinyl phosphate). We found the changes of pulse-pola-rographic properties of double stranded (ds) DNA after incubation with 01, especially at premelting temperature. There were differen-ces between DDVP and malathion, methylparathion and methylbromphen- vinphos. The structural changes caused by the latter three compounds were very insignificant. After thermal dénaturation of ds DNA pre-viously modified by 01 we observed the presence of thermolabile re-gions in DNA molecule.
INTRODUCTION
O rganoph osphorus insecticides belong to a group chemicals most widely used in agriculture. This is mos t due to the fact that though they are very effective as insecticides they do not last long in aquous environment, they are not accumulated in the soil and hence are no t den gerous for man. The examina tions of biolog ical activities of org anophosphor us insecticides (pi) in-dicate, however, that man y of them are harmful to living o r
-ganisms da ma gin g among others genetic mate rial of the cell. It was found that some 01 are carcinogenic ( tri chlo rfo n), te ratoge-nic (m al at hi on ), and some induce sister chromatids exch ange in the cultures of human lymphocytes (malathion, methylparathlon)[l, 2, 3, 4, 6], The y also show mu ta geni c activ ity similar to that of weak muta gene s and carcinogenes such as methyl methan sul phat e or d ichlorvos (DDVP) [5]. Our earlier experiments i ndica-ted that mala thi on added to human lymphocytes culture apart from evoking chromosomal aberrations significantly disturbs the m e ta -boli sm of n ucleic acids causing among others the changes of their synthesis rate and the decrease of DNA and RNA contents [7, 8].
Th e mech anisms of action of 01 on DNA have not been fully eluc idated yet. Alkyl ation of DNA by 01 ie surely one of the steps leading to its perma nent damage. The other type of action of 01 on DNA cannot be excluded i.e. the action of various, often very complicated, additional groups of particular 01. Though there exist a great numbe r of literature data concerning structural changes of DN A after the reaction with 01 they are mostly limi-ted to the presen tation of the chain breaks of DNA mo dified by 01 using diff erent methods (m ostl y centrifug ation in Cs Cl 2 or su-crose gradient) [9, 10, 11 ].
M oreover our previous paper showed that some subtle DNA d a -mages induced by 01 can also be investigated by puls-polarography m etho d [l2]. In this paper to mo d i fy DNA we used a few organo- phosphorus insecticides di fferent from each other not only in respect of their additional group but the structure of their c en-tral group as well. The obta ined results were to help us to d e -termine wh ether the classifi cation basing on their central group can be useful for the estimation of the harmf ulness of p ar t ic u-lar 01 as the agents dama ging DN A in vitro.
MATERIAL AND METHODS
DNA was isolated from calf thymus by Zamenhof's me thod [13 ]. The purity of DNA was checked by estima tion of RNA contents by Schneider's me thod [l4], protein content by Lowry's metho d [15 ],
and DNA content by B urton's m ethod [l6j. Particular values were as follows: <3%, <2%, >92%.
DNA was mod ified by the following standards of phosp horoór- ganic insecticides: malat hio n ( 0 , O-dimethyl/dic arboetoxyeth yl/ dithiopliosphate), DDVP (.0,0-dimethyl- 0-/dichlorov iny 1/phosp ha-te), met hylp ara thio n (0,0- dimethyl-0-/ 4-nitr oph enyl /t iono pho s- phate) and methy lbromph envi nph os (0,0-dlme thyl-0-l/2,4 -dichlo- ro/phe ny1-2-bromov inyl phosphate). DDVP and mala thio n wer e o b -tained from the Institute of Physical Ch emistry Polish Academy of Science in Warsaw, methyl parat hio n from the Institute of A p -plied Radiation Chem ist ry T echnical U niversity of Łódź and m e -thylbromphenvi nphos from the Institute of Organic Chem istry Tec h-nical Unive rsity of Łódź.
DNA at the concentr ation of 1 mg/ml in 0.1 M buffer Tris- HC1, ph 7.4 was incubated wit h particular 01 at the temperatu-re 37°C. Before adding to DNA insecticides wer e dissolved in 0.3 ml of 50% ethanol (on 10 ml of incubation mixture). The final concentrat ion of 01 in incubation mix tur e was 4.5 mM. After a certain time of incubation the reaction was stopped by pre cipitat ion of DNA with two volu mes of cold 96% ethanol. The DNA precipitate was diss olved in SSC/10 and dialysed ag a-inst SSC/10 for 20h. In experiments wit h de natu rat ed DN A the reaction was stooped by separation of the mo dy fi ng agent on S ephadex G-25 columns or by preci pitation of ss DNA with cold 96% ethanol plus 0.075 M M g C ^ .
All meas ure ment s were carried out by differential pulse-po- larography (d p p) metho d with an apparatus A 3100 Southern-Har- well Pulse Pola rograph (southern Ana lytical Ltd.) using me rcury dropping electrode. DPP experi ments were performed in a nitrogen atmosphere at the following settings: IV in 15 min. amplitude 50 mV, delay time 2 sec., amplifier sensivity 1/8 to 1/64 de- pendently cn DNA concentration. 0.3 M ammonium formate wit h 0.1 M sodium phosphate pH 6.9 or 5.0 was used as a background electrolyte. The principles and details of the polarogra phic meas urements were described by P a 1 e c e k et al. [l7, 18, 19]. Thermal denat uration was carried out by heating DNA sam-ples in SSC/10 to 1C0°C for 10 min., followed by quick coo l-ing in an ice bath. Alkaline denat uration of DNA was carried
out by M ixin g 0.S ml DN A in SSC/10 with 0.5 ml 0.2 M. NaOK; after 5 min. the solution was neutra lized wit h 1 M K H2PC>4 . Pre- mel tin g changes of DNA were performed by measu rin g the hight of peak II during heat ing the modi fied DNA from 20°C to 60°C.
RESULTS AND DISCUSSION
The results o btained in this paper confirmed our earlier reports that organopho sphorus insecticides react wi th DNA in vitro causing the damages of DNA native structure [l2]. As we used 01 from three diffe ren t classes it was possible to draw conclusions regarding the supposed me chanism of action of 01 with DNA. ‘
The first changes of pulse-p olar ogr aphi c properties of ds DNA are already observed a few hourn after the incubation with 01. The amount and character of these changes depend on the kind of insecticides. DDVP yields a small amount of ss DNA (peak Til) after a few hours of incubation while in case of oth er 01 it is po ssible to detect analogous amount of ss D NA only after 36 hours incubation. Earlier changes can be seen in peak II. This peak is formed at about 1.45 V i.e. 70 mV, mo re pos itive than peak III charact eristic for ss DNA. Peak II is charac teristic for ds DNA and is caused by mi no r local disturbances of h e-lical structure of DNA [l8, 1 9 The decrease of this peak a few hours after the reaction with methy lparathion, malathion and me thy lbromphe nvinphos can indicate the preferentialy action of the above men tioned compounds with these very regions. S i -milar effects were ob served during the reaction of DNA with sodium bisulfate whic h selectively induces the changes c onsi s-ting in deam inati on of p olarogr aphically active cytosine to no nred ucible uracyl [20, 2l]. It seems that in case of DNA m o -dified by 01 the decrease of peak II following alkylation is not only caused by the direct decrease of te amounts of n itr o-gen bases residues subject to polarog raphic reduction but also by other dist urbances effecting indirectly the DNA r eduction on electrode. They m ay among o thers consist in the local chan- !*js of the charge de nsit y in polynuc leotide chain and thus
in-c 1»
j
l
1 2 2 4 3 6 4 8 6 0 7 2 h o u rs F i g . 1 . T i m e c o u r s e o f t h e r e a c t i o n o f d o u b l e -s t r a n d e d D N A w i t h m a l a t h i o n A A , m e t h y l p a r a t h i o n o o , m e t h y l b r o m p h e n v i n p h o s O O , a n d D D V P • • a s m e a s u r e d b y m e a n s o f D D P i n 0 . 3 M a m m o n i u m f o r m a t e w i t h P 1 M a c e t a t e b u f f e r p H 5 . D N A c o n c e n t r a t i o n w a s 2 0 0 u e / m l . T h e p e a k h e i g h t o f t h e c o n t r o l s a m p l e w a s t a k e nfluence the adsorption' of a given polinucl eotide chain and its orientati on in the relation to negati vely charged electrode, and in turn they can inhibit ele ctroredu ction of poten tialy pola- ro graphi cally active bases residues. In case of DNA after the reaction with DDVP peak II cannot by mea sure d with sufficient occuracy since peak III wh ic h partly overlaps small peak II is formed very quickly (peak III is almost 100 times higher than peak II at the same DNA concentration). It is the same with other insecticides since during longer times of reaction with ds DNA peak III is so big that it is impossible to me asur e peak II of native DNA. For instance after 72 hours of reaction with malathion, met hylp ara thio n or methyl bromphe nvi nph os peak III corresponds to 2-3% of ss DNA in relation to thermally denatu- rated DNA as a sta'ndard. Nevertheless, the changes of pulse-po- larographic properties of native DNA after the reaction with 01 can be e xamined by carrying out the measurements at pH 5. Under such condit ions native DNA yields a character istic pulse-polaro- graphic peak, significantly higher than peak II formed in ne u -tral pH at the same DNA concentration. The potential of native DNA peak at pH 5 is almost identical to that of peak III of de natu ratod DNA. The measur ements at pH 5 confirm the observed differ ences in the kinetics of ds DNA between DDVP and other 01 (Fig. l). However, the appearance of some amount of ss DNA at pH 5 can interfere with the peak induced by other possible distur bances of dou ble- str and ed structure.
The structural changes of DNA caused by malathion, m et h y l -parathion and methyl bromphe nvi nph os are ve ry insignificant. It is confir med by the fact that in case of native DNA peak III appears onl y after 36 hours incubation with these insecticides. Earlier changes can be shown by pul se-pola rog raph ic analysis of ds DNA in neutral pH at premelting temperatures (30-60°c). Dp
to 35°C a small decrease of peak II is observed (Fig. 2). Sim
i-lar effe ct was noticed at neutral pH at room temperature while analysing the changes of peak II de pend ently on the reaction time (Fig. 3). Starting with 45°C peak II of DNA mo dified by 01 is significantly higher than the one formed by control DNA (Fig. 2). It seems that the pre sented differences are due to
Fig. 2. The dependence of the height of pulse-polarographic peak II on the temperature, ds DNA was incubated with malathion A---Û , methylparathion o----o , and raethylbromphenvinphos Q ----□ for the period of 6 h. DDP measure-ments were performed of a DNA concentration of 200 jig/ml. Background
elec-trolyte: 0.3 M ammonium formate with 0.1 M sodium phosphate pH 6.9
alkylation of rela tive ly small amount of n ucleotides both in case of the peak increase at pH 5 and the meas urements at pre- m elti ng temperatures above 40°C. The observed effect is most prob ably caused by the fact that in the nei ghbou rhood of m o -dified nucleo tides seme conformati onal changes of DNA take place w hi ch m ak e the undamage bases accesible for electrode processes. It is connected wi th the increase of p uls e-polar ographic peak. Similar changes were also seen after DNA irradiation wi th small doses of gamma radi ation [22], short dig estion with DNase I [ 2 3 ] and also in case of DNA alkylated to a small extent by MNU [24 ].
P ulse pola rography me asu rements carried out after thermal d é -n aturatio-n of ds DNA p rev ious ly m od ifi ed by 01 indicate the
Fig. 3. Time course of the reaction of native DNA with malathion Ô— — A , me-tliylparathion O---- O and methylbromphenvinphos □ --- □ as measured by means of DDP. DNA concentration 400 jig/ml. Background electrolyte: 0.3 M ammonium formate with 0.1 M sodium phosphate pH 6.9. The peak height of the control
samples was taken as 100%
pres ence of thermolabile regions in DNA mole cule (Fig. 4) while under analogous conditions of measure ments in alkaline solution such regi ons were not found. The depende nce of peak III of th er -mally dena tur ated DNA on the incubation time wi th OI (Fig. 2) is similar to that obta ined for ds DNA not subject to thermal dén atura tion (Fig. 1, 5). This fact suggests the existe nce of direct depen dence between the formation of a small amount of ss segments (about 2%) in ds DNA and the pres ence of thermolabile regions in molecule.
There are very few reports concerning the chemical nature or conformational changes induced by 01 in DNA. It is likely that guanine alkylation by 01 ma y lead to the appearance of ss
F i g . 4 . T h e d e p e n d e n c e o f t h e h e i g h t o f p e a k I I I o n t h e t i m e o f i n c u b a t i o n . D o u b l e -s t r a n d e d D N A w a s i n c u b a t e d w i t h m a l a t h i o n A A , m e t h y l p a r a t h i o n o O , m e t h y l b r o m p h e n v i n p h o s □ □ a n d D D V P • • f o r t h e p e r i o d i n d i c a t e d i n t h e g r a p h a n d s u b s e q u e n t l y d e n a t u r a t e d b y h e a t ( l 0 0 ° C , 6 m i n . ) . D D P m e a s u r e m e n t s o f d e n a t u r e d D N A w e r e p e r f o r m e d i n 0 . 3 M a m m o n i u m f o r m a t e w i t h 0 . 1 M s o d i u m p h o s p h a t e p K 6 . 9 a t r o o m t e m p e r a t u r e : c o n -c e n t r a t i o n o f t h e d e n a t u r e d D N A w a s 2 5 j u g / m l . T h e h e i g h t o f p e a k I I I a t z e r o t i m e i n c u b a t i o n w a s t a k e n a s 1 0 0 % ( p e a k h e i g h e r t h a n 1 0 0 % i n d i c a t e s a d e c r e a s e o f m o l e c u l a r w e i g h t o f t h e d e n a t u r e d D N A )
c-> F i g . 5 . F o r m a t i o n o f s i n g l e -s t r a n d e d D N A d u e t o t h e i n c u b a t i o n o f d o u b l e -s t r a n d e d D N A w i t h m a l a t h i o n A £ , m e t h y l p a r a t h i o n o O , m e t h y l b r o m p h e n v i n p h o s □ □ a n d D D V P • « a s d e t e c t e d b y : p o a n s o f D D P . D D P m e a s u r e m e n t s w e r e p e r f o r m e d a t a D N A c o n c e n t r a t i o n o f 4 0 0 > i g / m l i n 0 . 3 M a m m o n i u m f o r m a t e w i t h 0 . 1 M s o d i u m p h o s p h a t e p H 6 . 9
reyions in the neighbourho od of modifi ed bases similar to the reaction on DNA wit h N-acetoxy-N-2-acetylair.inofluoren [25, 26 J. Many papers state that d epurinated DNA is rather easily h y dr o-lyzed in alkaline solutions while alkylated DNA itself is u n -der these conditions very stable. Alky lated purines and apuri- ne sites are easi ly hydroly zed at eleva ted temperature what leads to the breaks of polynuc leotide chain. Depuri nation is an intermediate step in the thermal de gra dation of alkylated DNA [27]. It can be supposed that the mo dif ication of DNA by 01 leads to the labilisation of phosph o-sugar chain. At elevated temperature the demin ish ed stability can be seen due to the breaks of ph ospho-s ugar chain at the damaged regions. In case of ss DNA it is accompanied by the decrease of its molecular weight what is observed on pulse-polarograrr.s as the increase of peak III of thermally de natur ated DNA (Fig. 2). The comp a-rison of the reaction kinetics of particular 01 w it h DNA shows how di fficult it is, basing onl y on chemical composition of a given compound, to determine its pot ential harmfulness in in-ducing DNA damages. Among the investigated insecticides DDVP (phosphate) is the compound which to the largest extent effects tht electroch emical properties of DNA. However, the kinetics of the reaction of methylbr ophe nvln pho s (also phosphate) with DNA resambles rather the one obtained for mala thi on (dithiopho- sphate) or methylp arat hio n (t iono ph os ph at e) . It seems im proba-ble, therefore, thet the extent of DNA damages bv partic ular 01 depends first of all on the structure of their central group. This process appears to be far more comp licated and its e lu -cidation requires further intensive investigations.
Independently of the obtained results the present paper c on -firmed the usefulness of puls e-polarograp hy method in examining subtle changes of DNA structure evok ed by vari ous factors re ac -ting with DNA. In our earlier paper we reported that 72h in-cubation with malat hion dose not cause any changes in DNA struc -ture wh ic h could be noted using the temperature me ltin g curve metho d or fractionation on hydroxyapatite [12], Using pulse-po- larograpny m etho d we found significant changes of el ect ro ch em i-cal properties of DNA oven after shorter time of reaction.
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Department of Molecular Genetics University of Łódź, Poland
Ryszard Wiaderkieiri.cz, Zofia Walter
CHARAKTERYSTYKA PULS-POLAROGRAFICZNA DNA
ZMODYFIKOWANEGO INSEKTYCYDAMI FOSFOROORGANICZNYMI IN VITRO
Przeprowadzono badania metodą puls-polarograficzną DNA izolowanego z gra-sicy cielęcia i zmodyfikowanego związkami fosforoorganicznymi. Zbadano wpływ następujących związków: malationu (0,0-dimetylo-S-1-2-dikarboetoksyetyloditio- fosforanu), DDVP (0,0-dimetylo-0-1-2-dichlorowinylofosforanu), metyloparatio- nu (0,0-dimetylo-0-4-nitrofenylotionofosforanu) i metylobromfenwinfosu (0,0-di- metylo-0-1-2,4-dichlorofenylo-2-bromowinylofosforanu). Zaobserwowano zmiany własności pulspolarograficznych po inkubacji dwuniciowego DNA ze związkami fosforoorganicznymi. Zmiany dotyczyły szczytu II i III. Szczyt II jest cha-rakterystyczny dla dwuniciowego DNA i jest wywołany niewielkimi zaburzeniami helikalnej struktury. Szczyt III jest charakterystyczny dla jednoniciowego DNA. Po 72-godzinnej inkubacji z malationem, metyloparationem lub metylobrom- fenwinfosem szczyt III odpowiadał 2-3% zawartości jednoniciowego DNA w odnie-sieniu do zdenaturowanego termicznie DNA jako wzorca. Pomiary w pH 5 wykazały
różnice w kinetyce zmian dwuniciowego DNA między DDVP i innymi badanymi in-sektycydami. Zmiany strukturalne DNA wywołane przez malation, metyloparation i metylobromfenwinfos były bardzo nieznaczne. Obserwowano zmiany w zakresie temperatur przedtopnieniowych (30-60°c). Polarograficzne pomiary przeprowa-dzone po termicznej denaturacji dwuniciowego DNA, zmodyfikowanego uprzednio związkami fosforoorganicznymi, wykazywały obecność regionów termolabilnych.
