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
FO LIA B IO C H IM ICA ET BIO PH Y SICA 13, 1998
Joanna Kowalik, Janusz Błasiak
COM PARISON OF THE DNA DAMAGING ACTIVITY OF THE ORGANOPHOSPHORUS
INSECTICIDE METHYLPARATHION AND ITS MAIN METABOLITE
The ability o f the comm only used organophosphorus insecticide m ethylparathion and its main metabolite m ethylparaoxon to induce dam age to pU C19 plasm id D N A was investigated. Agarose gel electrophoresis was employed in the study. T he changes in optica] density o f bands corresponding to supercoiled (CCC) and open circular (OC) form o f pU C19 were analysed. M ethylparathion, in contrary to its m etabolite, did n o t cause any changes as detected by the used system. M ethylparaoxon evoked conform ational changes o f the D N A th a t were displayed as the increase o f the fraction o f the O C form. Obtained results indicate that m ethylparaoxon has potential ability to induce dam age to D N A in vivo.
1. IN TRO D U C TIO N
Organophosphorus insecticides are used in agriculture and food production. They are characterised by high efficiency and low persistence [1], The organophosphorus insecticides are powerful inhibitors o f acetylcholinesterase. This target is responsible for m ost o f the physiological effects, e.g. hyperex citability, convulsions and m uscular paralysis, events which precede death in poisoned animals [2-4], Additionally, other m arkers o f toxicity (memory and visual disturbances, schizophrenia and depression) not related to the inhibition o f acetylcholinesterase m ay chronically develop in poisoned individuals [5-7],
Chronic toxicity in m an is related to behavioral, genetic, reproductive, teratogenic and carcinogenic actions [5], but the m olecular m echanism s of these effects are far from clear. Therefore, the knowledge of the interactions of insecticide com pounds with biomolecules is an imperative task for the design and synthesis of selective compounds.
If the genotoxicity of a com pounds is concerned, it is essential to assess the ability of the com pounds to induce D N A damage. To do so an in vitro system which looks at the effect o f the insecticides on purified D N A
directly can be used [8], This system is organism independent and is free o f the m asking DN A repair and is also free of any m etabolic capacity, e.g. in terms o f bioactivation and detoxification. M oreover, this system allows to interpret obtained results in relative precise physico-chemical terms.
Plasmid D N A is a useful tool for the investigating of dam ages to DN A. The plasmid D N A can be in three conform ational forms differing in electrophoretic mobility in agarose gel. Native plasmid represents superhelical, covalently closed form (CCC). Single-strand breaks lead to relaxation of plasm id D N A and generation o f open circular form (OC). Covalent intercalation between base pairs can cause the same result. D ouble-strand breaks induce formation of linear form. D N A nicking agents and intercalating substances in vivo can lead to genotoxic changes that can be displayed in the electrophoretic pattern of plasmid DN A.
Possibility to induce damage to plasmid D N A by the organophosphorus insecticide m ethylparathion and its m ain m etabolite m ethylparaoxon was investigated in this work.
2. M A TERIA LS AND M E T H O D S
D N A
pUC19 plasmid D N A was obtained from Peterfarm (Sieradz, Poland). The concentration o f D N A was estimated spectrophotom etrically. Plasmid D N A typically contained about 80% double stranded covalently closed circular supercoiled molecules, 20% open relaxed circular molecules and no linear molecules.
Chemicals
O rganophosphorus com pounds m ethylparathion (0 ,0 -d im eth y l 0 -4 nit- rophenyl phosphorothioate) and its m ain m etabolite m ethylparaoxon ( 0 ,0 - dimethyl 0 -4 nitrophenyl phosphate) at purity o f 95-99% were supplied by Instytut Przemysłu Organicznego (Warsaw, Poland) and D r. Ehrenstorfer G m bH (Augsburg, Germany). Chemical structures o f these agents are displayed in Fig. 1.
s o
( C H p ) 2P - 0 - ^ ^ N 0 2 ^ ' n C>( C H f l ) 2P - 0 - ^ ^ N 0 2
a b
Chemicals treatment
The insecticide and its m etabolite were derived from stock (50 m M ) ethanolic solutions to give a final concentration o f 150 /¿M. The D N A and ethanol concentration in all samples were respectively 13,3 //g/ml and 0.384% . T he control received, instead o f the insecticide, ethanol, the concentration o f which did not affect the processes under study. The samples were incubated in the dark for 72 h at 37° C in a buffer comprising 45 mM Tris-borate, 1 mM ED TA , pH 8.0.
Sample analysis
Covalently closed circular D N A (CCC), such as the pU C19 plasmid used in this experiment, is a sensitive probe for detecting strand scis sion [9]. Such plasmid can be cleaved at any of phosphodiester linkages which permits the D N A strands to unwind, resulting in relaxed form OC D N A [10]. Introduction of another nick adjacent to the first but on the opposite strand gives linear form DN A. Some chemicals m ay not only cause D N A strand breakage but also unwinding of negatively supercoiled D N A by intercalative covalent m odification [11-13]. Both processes lead to a form ation o f a relaxed circle which agarose gel electrophoretic m obi lity is about half that of the supercoil. The samples were ran in 0.8% agarose, stained with ethidium bromide, placed in a UV transillum inator and photographed with a Polaroid cam era using a black and white Pola roid film type 665. The negatives were scanned with a D esaga densitom e ter, m odel CD 60.
3. RESULTS
Result o f agarose gel electrophoresis of pUC19 plasmid D N A after 72 h incubation with m ethylparathion and m ethylparaoxon at a concentration of 150 //M is displayed in Fig. 2. The control (lane 1) shows three bands corresponding to D N A s o f different electrophoretic mobilities. The first one o f high optical density, m igrating through agarose gel at highest rate, represents native supercoiled plasmid D N A (CCC). The second, slower m igrating band, corresponds to relaxed circular form (OC). The third visible band in the electrophoretic pattern is chromosomal D N A contaminating plasmid D N A .
: i : V , Ï H M l g f M I I j .\v' i m v ; w : ,: . i:r- ■ -'i - : ' ! > • i ■ - : ' s " . : '. J k V s » /A > ■ Î * * * >, § ■ ? - < > > • « ■ y ^ p g B I - j • ‘ r » ■ •»(>!*< *u>vuvv
,t
'Æ jjj1
,;'VFig. 2. Electrophoresis of plasmid pU C19 D N A after 72 h incubation a t 37° C w ith methyl- p arathion (lane 2) and m ethylparaoxon (lane 3) a t a concentration o f 150 /¿M. Lane 1 is the control. 100 ng o f D N A was loaded on each slot o f 0.8% agarose gel and electrophoresis
was performed a t 5 V/cm for 3 h
Com paring to control sample, no differences were observed in electro phoretic pattern after incubation of plasmid pUC19 D N A with methyl- parathion (lane 2). Increase o f optical density of the band of OC form and decrease of optical density o f CCC form band followed after incubation of plasmid D N A with m ethylparaoxon.
4. D ISC U SSIO N
C om parison of samples treated with m ethylparaoxon with control shows the differences in optical density of bands corresponding with superhelical (CCC) and open circular (OC) forms o f plasmid D N A . Therefore, m ethyl paraoxon has an ability to induce conform ational transform ation of studied
D N A by disturbing its superhelical structure. This can be a consequence o f single-strand breaks or intercalative covalent m odification. Performed experiment does not allow to differentiate which of these two mechanisms participate in observed transition of CCC into OC form.
M ethylparathion is metabolised to m ethylparaoxon in organism [14] - the phosphorothioate residue is oxidized to phosphate residue (Fig. 2). The properties of natural m etabolites should be considered during studies of the influence of a chemical on living organism.
Oxidation is a typical reaction o f organophosphorus com pounds [5]. It takes place in m icrosom al fraction of hepatocites and has influence on biological activity o f these compounds. In general, oxidative analogues of organophosphorus insecticides are m ore toxic than parent com pounds [5], w hat was confirmed in our experiment. D ouble bond between phosphorus and oxygen atoms causes that the former one acquires electrophilic properties. It can cause an attack of insecticide molecule on nucleophilic com pounds, such as D N A .
Possible mechanism leading to D N A strand nicking after organophos phorus insecticide treatm ent is m ethylation of purines and spontaneous depurinization of alkilated bases. U nder physiological conditions (pH 7.4; 37° C) the apurinic sites are unstable and D N A is cleaved by a /^elim ination mechanism [15],
F act that m ethylparaoxon has an ability to interact directly with D N A generating strand breaks of purified D N A or intercalating between base pairs m ay not necessarily be a basis of reported genotoxic properties of this com pound or inducing identical effects in vivo. F u rth e r studies are needed to establish possible mechanism of observed changes.
5. REFEREN CES
[1] W a r e G . W. (1978), The Pesticide Book, vol. I, W. H. Freem an and Com pany, San Francisco, 27-52.
[2] A l d r i d g e A. N., D a v i s o n A. N . (1952), Biochem. J., 51, 61. [3] D o h e r t y J. D . (1979), Pharm acol. Ther., 7, 123.
[4] F u k u t o R. T. (1990, Environ. H ealth Perspec., 87, 245.
[5] E t o M . (1974), Organophosphorus Pesticides: Organic and Biological Chemistry, CRC Press, Cleveland, 123-231.
[6] O k h a w a H., O s h i t a H., M i y a m o t o J. (1980), Biochem. Pharm acol. 29, 2721. [7] H e r b e r t G., P e t e r l e T . , G r u u b T . (1989). Bull. Environ. Contam. Toxicol., 42, 471. [8] G r i f f i n D. E., H i l l W. E. (1978), M utat. Res. 52, 161.
[9] G a m p e r H. B., T u n g A. S. C., S t r a u b K. , B a r t h o l o m e w J. C., C a l v i n M. (1977), Science, 197, 671.
[10] J o h n s o n P. H. , G r o s s m a n L. I. (1977), Biochemistry, 16, 4217. [11] E s p e j o R. T., L e b o w i t z J. A. (1976), Anal. Biochem., 72, 95. [12] B r a w n K. , F r i d o v i c h I. (1981), Arch. Biochem. Biophys., 206, 414.
[13] S a l g o M . G ., S t o n e K., S q u a d r i t o G. L., B a t t i s t a J. R., P r y o r W. A. (1995), Biochim. Biophys. Res. Comm un., 210, 1025.
[14] M a t s u m u r a F. (1985), in: Toxicology o f Insecticides, ed. F. M atsum ura, vol. 2, Plenum Press, N ew Y ork and L ondon, 203-289.
[15] K u s h i d a T. (1994), J. Am. Chem. Soc., 116, 479.
Cam e in editorial office D epartm ent of M olecular
„F olia biochimica et biophysica” Genetics, U niversity o f Łódź
11.12.1996 r. Poland
Joanna Kowalik, Janusz Błasiak
PO R Ó W N A N IE Z D O L N O Ś C I DO U SZK AD ZAN IA DNA
P R Z E Z INSEKTYCYD FO SFO R O O R G A N ICZN Y I JE G O G ŁÓ W N Y M E T A B O LIT
Badano zdolność indukow ania uszkodzeń D N A plazm idu pU C19 przez powszechnie stosowany insektycyd fosforoorganiczny metyloparation i jego główny m etabolit metyloparaokson p o 72 h inkubacji z tymi związkami. Stosowano metodę elektroforezy w żelu agarozowym i analizow ano zmianę intensywności pasm odpowiadających dw óm form om konform acyjnym plazm idu - kolistej superskręconej (CCC) i kolistej otwartej (OC). M etyloparation, w przeci wieństwie do swego m etabolitu, nie wywoływał zmian konform acyjnych plazm idow ego D N A . D ziałanie m etyloparaoksonu pow odow ało przejście konform acyjne D N A z form y CC C d o OC. O trzym ane rezultaty wskazują na potencjalną zdolność m etyloparaoksonu do wywoływania uszkodzeń D N A in vivo.
