A C T A U N I V E R S I T A T 1 S L O D Z I E N S I S
F O L IA B IO C H 1 M IC A E T B IO P H Y S IC A 9, 1992
Janusz Błasiak, ZoJ'ia Walter, M aria Gawrońska
E F F E C T S O F S O M E O R G A N O P H O S P H O R U S IN S E C T IC ID E S O N C A L C IU M , P O TA SS IU M
A N D C H L O R ID E E F F L U X F R O M IS O LA TE D P IG L Y M P H O C Y T E S
U sin g a n is otop e labe ling te ch niqu e, we inves tiga ted the release o f p o tas sium , ca lcium , a nd c hlo ride ions from pig ly m phocytes in vitro u n d e r influe nce o f som e o rg a n o p h o s p h o ru s c o m p o u nd s used a s insecticides. T w o o f used a gents, viz. m ethyl p a ra th io n a n d m elh y l-b rom p h e n vin p h os p ro du c ed sig n ific an t (o fte n exce eding 2 0% ) ch an ge s in the q u a n tity o f the efflux o f all tes ted ions, while the re m a ining two: m a la th io n an d d ic hlorv os ch ang ed the efflux in lesser degree. T he ph en om eno log ica l c h a ra c te r o f exp erim e nt did n ot allow to d ra w con clu sion c o n ce rn in g the m e ch anism o f obse rved change s. T h e ob ta in ed results ex tend on som e ions, o u r p rev io us findin g c on ce rn ing ch ange s in tra n s p o rt o f nucleic acids an d D N A /R N A m odifyin g enzym es th ro u g h ly m p hoc ytes m em bran e cau sed by o rg a n o p h o sp h a te insecticides.
1. IN T R O D U C T IO N
O rg an o p h o sp h oru s insecticide are widely used in agricu ltural p ro du ction . D ue to their low w ater solubility they can interact with structu res which are rich in lipids, such as biological m em branes. It was confirm ed by the results o f experim ents in which th ere were observed changes in activity o f m em b rane b ou nd enzymes [5, 9, 13, 14, 18], q uan titativ e changes in lipid fractio n [7, 10, 12], changes in fluidity and m em b rane ch arg e [4, 9, 11], changes in p er-m eability for electrolytes [2, 3] and nonelectrolytes [1, 2] u nd er the influence o f o rg a n o p h o sp ho ru s insecticides.
P reviously we showed th a t the lym phocytes released an increased am ou n t o f nucleic acids an d D N A /R N A m odifying enzym es into the ex tracellular environ m ent, follow ing in cub ation with insecticide m alath ion . The observed changes were associated w ith depolim erization o f nuclear D N A and va riatio n o f am o u n t o f D N A in subcellular fractions [21, 22], The m echanism underlying these p hen o m en a is un kn ow n yet, b u t the results o f research on influence o f
insecticides on biological m em b ranes suggest th a t the changes m ay be asso ciated w ith d isturban ces o f m em bran e tra n sp o r t processes.
In this w ork we try to check w hether o rg an o p h o sp h o ru s insecticide can affect also tran sp o r t o f p otassium , calcium and chlo rid e ions thro u gh the lym phocyte m em bran e.
2. M A T E R IA L S A N D M E T H O D S
P orcin e blood was o btain ed from a local slaughtery. P eripheral blood lym phocytes were isolated acco rding to the m etho d o f P ark es an d Bradley [20], The lym phocytes were suspended at a co nc en tration o f l()x cells per ml in a p h o sp h ate saline buffer (PBS) a t pH 7.4. Cell viability was estim ated by the ab ility o f the cell p o pu latio n s to exclude T ryp an Blue an d was never less th an 95% .
U sing isotop e labeling technique we investigated the efflux o f potassium (86R b + ), calcium (45C a2 + ), an d chloride (36C1 ) from the isolated lym phocytes in the presence o f vario us o rg an o p h o sph o ru s insecticides: m ethy l-b rom ph en - vin phos (2-b ro m o -l-(dich loro ph eny l)vin yl dim ethyl p ho sph ate), dich lorvos (2,2-dichlorovinyl dim ethyl ph o sp h ate), m alath io n (S -1,2-bis-(ethoxycarbo- nyl)ethyl O -O -dim ethyl p h o sp h o ro th io a te, and m ethyl p ara th io n (0 ,0 -dim et- hyl O -p -nith ro p heny l p ho sph o rothio ate). Investigations were carried o ut at three final co nc entra tio n s o f each insecticide: 10 5, 10 4 an d 10 3 M. The insecticides were derived from co n cen trated (50 m M ) so lutio ns in ethan ol. Th e co n tro l g rou p received only eth ano l at a co nc entration ap p r o p r ia te fo r the m axim al co n cen tratio n o f the p artic ular insecticide (never m ore than 0.92% ). O ne g ro u p got n either an y insecticide n or ethanol.
Each isotope w as add ed to the cells su spended in PBS a t a co n ce ntratio n o f 108 p er ml in such a m o u n t th at the final specific activity w as 2 /¿Ci/ml for 86R b and 45C a and 1 n Ci/m l for 36C1. T he lym phocytes were irçcubated for 1 h at 37°C in the presence o f a p ar tic u lar isotope, cooled to 0°C an d w ashed fo u r tim es in ice-cooled PBS; each w ashing was followed by 10 s cen trifug atio n at 10,000 g. 0.3 ml aliq u o t o f the lym phocyte suspension was collected in small tubes, each c on tain in g 1 ml PBS an d an insecticide a t a desired con centratio n. A fter 1 h releasing an isoto pe a t ro om tem p eratu re, the sam ples were cen trifug ed a t 10,000 g for 30 s, 0.5 ml aliq uo t o f su p er n ata n t was ta ken , m ixed w ith 3 ml o f scintillation cocktail and counted in a scin tillato r c o un ter (LK B W allac R ack B eta 1209) to estim ate th e a m o u n t o f radioactiv e isotop e released by the lym phocytes. Ten tho u san d cou nts were collected for each p ro b e to ensure a sta n d a rd deviation o f ± 100 co un ts o r 1%. T he scin tillato r co u nter au tom atically conv erted th e cou nts, w ith a correctio n for dead time, to cou nts p er m in ute (cpm).
Each exp erim ent w as perform ed in 10 replications. The average rad io a c-tivity in the g rou p which received neith er any insecticide n o r eth an o l was used to norm alize the radio activity in the rem aining groups. A q u an tity o b tain ed in this way was fu rth e r called „efflux” . O ne-w ay analysis o f variance was used to test for effects o f insecticides.
3. R E SU L T S A N D D IS C U S SIO N
T ab le 1 gives the results o f efflux o f S6R b + ions from the lym phocytes. At all tested co n cen tratio ns o f m ethyl-bro m p hen vin ph os a statistically significant en hanced efflux o f 86R b + w as observed. Th e enh ancem ent rang ed from 20% (10 5 M ) to nearly 30% (10 3 M). An enhancem ent o f efflux caused by dichlo rvo s was ind epen den t o f its co nc en tratio n and averaged 6% . M alath ion had no statistically significant effect on the release o f WlR b + . M ethyl p a rath io n pro du ced a decrease in the releasing o f 8fiR b + at all tested co n centratio ns. T he decrease ranged from 21% (10 5 M ) to 16% (10 3 M).
T a b l e 1
E fflux o f 86R b + from ly m p ho cy te s u n de r influence o f o rg a n o p h o s p h o ru s insecticides (m ean ± SD , n = 10, all exps.). F o r the e xp la n a tio n o f th e term „ efflu x” see M a teria l a nd M e th o d s
Insecticide C o n c e n tra -tion [M] E fflux P* M e th y l-b ro m p h en v in p h o s 0 I0 5 10 4 10 3 1.0007 ± 0.0529 1.2041 ± 0.2023 1.2738 ± 0.1516 1.2997 ± 0.1296 <0 .0 1 <0 . 0 0 1 <0 . 0 0 1 0 0.9666 ± 0.0209 10 5 1.0261 + 0.0326 <0 . 0 0 1 D ic h lorvo s 1 0 4 1.0193 ± 0.0441 < 0 .0 0 5 1 0 3 1.0318 ± 0.0405 <0 . 0 0 1 0 0.9629 ± 0.0361 10 5 0.9812 + 0.0160 >0.1 M a la th io n 1 0 4 0.9540 ± 0.0202 > 0 .5 10 3 0.9768 ± 0.0209 >0 . 2 0 1.0213 ± 0.0665 10 5 0.8055 + 0.0397 <0 . 0 0 1 M eth yl p a ra th io n 104 0.8272 ± 0.0323 <0 . 0 0 1 1 0 3 0.8594 ± 0.0704 <0 . 0 0 1
T he exposu re o f th e lym phocytes to m eth yl-b ro m p h en vinp ho s at co n cen t-ratio n s 10 4 and 10 3 M pro du ced a significant redu ctio n o f calcium efflux (9 and 12% respectively) - see Table 2. D ichlo rvo s did n o t cause any statistically significant chang es bu t m alath ion did a 10% enhancem ent at 10 3 M. M ethyl p ar a th io n p ro du ced an enh ancem ent in the releasing o f calcium a t co n cen t-ratio n s 10 4 M (3 0% ) and 10" 3 M (28% ).
T a b l e 2
El'll ux o f 45C a 2+ from ly m pho cytes u n d e r influence o f o rg a n o p h o s p h o ru s insecticides (m e an ± SD , n = 10, all exps.). F o r the e x p la n a tio n o f the term „e fflu x " see M a terial an d M eth o ds
Insecticide C o n c e n tra -tion [M] E fflux P* 0 0.9822 ± 0.0462 M e th yl-b ro m p h en v inp h o s 10 5 104 0.9520 0.8977 ± ± 0.0633 0.0305 >0 . 2 <0 . 0 0 1 10 3 0.8617 ± 0.0451 <0 . 0 0 1 0 1.0070 ± 0.0615 D ichlo rvo s 10 5 0.9646 ± 0.1003 >0 . 2 104 1.0456 + 0.0719 >0 . 2 10 3 0.9859 ± 0.0713 > 0 .5 0 0.9804 + 0.0689 M a la th io n 10 5 0.9775 ± 0.0926 > 0 .5 104 1.0175 ± 0.1489 >0 . 2 10 3 1.1641 ± 0.1434 <0 . 0 0 2 0 0.9901 + 0.1108 M eth yl p a ra th io n 10 5 104 1.0454 1.2910 ± + 0.1691 0.1777 >0 . 2 <0 .0 0 1 10 3 1.2688 ± 0.1833 <0 .0 0 1
* Scheffe’s m ultip le c o m p a ris o n m e th od .
The ex posure o f th e lym phocytes to m ethy l-b ro m p h en vinp ho s resulted in dim inishing o f efflux o f chloride at co nc entratio n s 10 4 M (8% ) an d 10 3 M (10% ) - see T ab le 3. D ichlorvos did n o t affect the efflux at all tested co n cen tratio n s. M alathion caused enhan ced efflux a t 10 4 M (11% ) an d 10 5 M (13% ). M ethyl p a ra th io n p ro du ced a significant decrease in th e efflux a t all con cen tratio ns rang in g from 12% at 10 3 to 24% a t 10~5 M.
The o b tained results testify to changes in tra n sp o rt o f p otassium , calcium and chloride ions caused by som e o rg an o p h o sp h o rus co m po u n ds used as insecticides. T he n a tu re o f th e p erform ed stud y does n o t allow to draw co nclusions o n th e m echanism (s) w hich m ay underlie the ob serv ed changes.
T here are a n u m b er o f conflicting results o f investigations dealin g with insecticides and tra n sp o rt o f ions. The o rg a n o p h o sph o ru s insecticides p ara- th io n, azinp ho s an d m alath io n proved to be activ ato rs o f ra b b it sarcop lasm ic reticulum (C a 2+ + M g2 + )-A TP ase and increased the ratio o f translo cated C a 2+ to hydrolyzed A T P [3], On the o th er han d m alath ion was show n to be an in hib ito r b oth o f (C a2+ + M g2+ )-A T P ase activity an d A T P -dep end en t calcium tra n sp o r t in b rain sy napto so m e m em bran e vesicles [17]. It w as also show n th a t som e o rg an o p h o sp h ates had an inh ibito ry effect on the activity o f ( N a + , K + )-A T P ase an d (C a2+ + M g2 + )-A T Pase o f ra t h ea rt [8] an d (N a + , K + )-A TP ase o f ery thro cy te m em b rane [15].
T a b l e 3
Efflux o f 36C1 from ly m p ho cy te s u n d er influe nce o f o rg a n o p h o s p h o ru s inse cticides (m e an ± SD , n = 10, all exps.). F o r the e x p lan a tio n o f th e term „ efflu x" see M ateria l a n d M e th o d s
Insecticide C o n c e n tra -tio n [M] Efflux P* M e th y l-b ro m p h e n v inp h o s 0 10 5 10 4 10 3 0.9408 ± 0.0834 0.9343 ± 0.0863 0.8673 ± 0.0613 0.8492 ± 0.0787 > 0 .5 < 0 .0 5 < 0 .0 5 0 0.9432 ± 0.1125 10 5 0.9404 + 0.1099 > 0 .5 D ic h lorvo s 10 4 0.9302 + 0.1274 > 0 .5 10 3 0.8492 ± 0.8883 >0 . 2 0 1.0032 ± 0.0799 10 5 1.0434 + 0.0526 >0 . 2 M ala th io n 10 4 1.1155 ± 0.0683 < 0 .0 0 5 10 3 1.1312 ± 0.1030 <0 .0 1 0 1.0254 ± 0.1111 io -5 0.7755 + 0.0566 <0 .0 0 1 M ethy l p a ra th io n 10 4 0.8596 ± 0.0915 <0 . 0 0 2 1 0 * 3 0.9056 ± 0.1318 < 0 .0 5
* Sch effe's m ultip le co m p a ris o n m etho d.
T he insecticides m ala th io n an d p ar ath io n exert significant effects on the io n o p h o re - indu ced perm eability o f liposom e m em branes for K r an d Ca~ + ions as a consequence o f changes in th e fluidity o f m em bran e lipids [2],
T he base o f biological activity o f insecticides sho uld be considered on the m olecular level. Th e stru ctu re o f atom s P-O -C which is typical of o rg a n o p h o s-p hates seems to be fu nd am en tal as far as the biological action o f these co m po u n ds is co nsidered. This stru cture is m ainly responsible for in teraction
o f o rg an o p h o sp h a te w ith nucleophiles o f the cell. N ucleic acids and pro teins have the largest am o u n t o f nucleophilic sites o f the cell. A nucleop hile can preferably attac k a p h o sp h o ru s ato m causing the break o f the -P-C - b on d an d, in tu r n , p h o sph o rylatio n , o r b reak s -C-O- bon d causin g m éthy latio n. An inh ibitio n o f the active site o f acety lcholinoesterase is the best know n m éthylatio n reactio n caused by insecticides [6, 16, 23]. O rg an o p h o sp ha tes can cause m éthy latio n o f p rotein s [23] followed by in hib itio n o f N a + /C a 2 f exchange, being a com p o n en t o f the calcium tra n sp o rt system o f biological m em branes.
D ue to the great im po rtance o f ion tra n sp o rt processes in the cell and lack o f agreem ent in assessing the influence o f o rg an o p h o sp h o ru s agen ts on these processes the u n d erta ken investigations need fur th er study.
Acknowledgements
This w ork was su p p o rted by the M inistry o f N a tio n al E du cation Research P ro gram PR II 11.4.11.
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C a m e in e d ito ria l office D ep artm en t o f M o le cu la r G ene tics „ F o lia b ioc him ic a et b io p hy sica ” In s titu te o f B iochem istry
25.05.1991 U niv ersity o f Ł ó dź
J anu sz B łasiak, Z o fia W aller. M aria Gawrońska
W PŁ Y W IN S E K T Y C Y D Ó W F O S F O R O O R G A N IC Z N Y C H N A W Y P Ł Y W W A PN IA , P O T A S U I C H L O R U Z IZ O L O W A N Y C H L IM F O C Y T Ó W Ś W IN I .
B a da no , s to sują c te chn ik ę z n ak o w a n ia izo top o w e go , u w a lnia nie jo n ó w p o ta s u , w ap n ia i c h lo ru z lim fo cytó w świni in vitro p od wpływ em zw iąz ków fos fo ro orga nicz ny ch używ anyc h ja k o insektyc ydy. D w a s p o ś ró d s to so w any c h zw iązków , m ianow icie m e ty lo p a ra tio n i m e ty lo brom fen - w infos p ow o d ow ały z nac zące (często p rzek ra cza ją ce 2 0% ) zm ia ny w wielkości w ypły w u, po dc zas gdy p oz o stałe dwa: m a la tio n i d ic hlorfo s o ddz iaływ ały na wypływ w m niejszym s to pn iu . F en om en olog ic zn y c h a ra k te r p rz ep ro w a d z o ne go e k sp ery m e ntu nie po zw ala ł n a sform ułow a nie w nioskó w d otyc ząc yc h m ech an iz m ó w leżących u p o d staw o bse rw o w an yc h zm ian. O trz ym a n e re zu lta ty s ta no w ią rozs zerzen ie o n ie które jo n y wcześniejszych stw ierd zeń d otyc ząc yc h z m ian w tra n sp o rc ie kw asó w nu kleino w yc h i enzy m ów n ukleo lity cznyc h p op rz ez błony lim focy tów po d w pływ em in sek tyc ydó w fos fo roo rg an icz ny ch .
