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
F O L IA B IO C H 1 M IC A E T B IO P H Y S IC A 14, 1999
Z o fia W alter, M aria Gawrońska, Christos Kargas, Wanda M ajcherek
EFFECT OF M ETHYLPARATfflON ON GLUTATHIONE S-TRANSFERASE ISOENZYMES IN DIFFERENT RAT TISSUES
In th is w o rk we exam ined the influence o f the o rg a n o p h o sp h o ro u s insecticide (IF O ) m e th y lp a ra th io n o n th e activ ity o f g lu ta th io n e S -tra n sfera se in vivo in d ifferen t m ale W istar r a t tissues. W e exam ined p o stm ito c h o n d ria l, cytosol, and m icro so m al cell fractio n s, o f the liver, kidney, p an creas, spleen, and b ra in . T h e activity o f the G S T w as m easu red acco rd in g to each o f th e tw o su b strates: C D N B an d D C N B respectively, w h at p e rm itte d us to d iffere n tia te G S T isoenzym s. W e observed significant increases o f th e activity o f G S T w hich d e p en d ed o n the tissue an d cell fractio n .
IN T R O D U C T IO N
P h o sp h o ro -o rg a n ic co m p o u n d s used as insecticides (IF O ) con siderably d istu rb cell m etab olism in near-lethal doses to w hich m am m als are generally exposed in consequence o f using IF O in agriculture and in households.
IF O dam ag e deoxyribonucleic acid and d istu rb th e tran sfer o f genetic in fo rm atio n [2-4, 24-26, 28-32]. In vivo they pro duce m u ta tio n s and they also can be carcinogenic and teratogenic factors (6). T h e m echanism which leads to such serious dam ages is n o t fully k now n yet, because the d a ta in investigations in vivo relating to dam ages to genetic m aterials are d isco rd a n t an d n o t uni vocal.
T his p ap e r has been devoted to the investigation o f th e influence o f m eth y lp ara th io n (M P) on glutathione S-transferases o f different cell fraction s o f ra t liver, kidney, spleen, pancreas and brain. T he m etab o lism o f M P in ra t liver has been presented in Fig. 1 [10].
M e th y lp a ra th io n is activated by d esulfurization to p a ra o x o n (in h ib ito r o f acetocholinoesterases in m icrosom al fractio n o f th e liver). E n zy m atic
CH,0 S \ 0 p / X o CH30 0
CH3° \
/ (0)
n o2 n o2Fig. 1. M e tab o lic p a th w a y o f m ethyl p a ra th io n in r a t liver
1 - m icro so m al m ix ed -fu n ctio n oxidases, 2, 3 - g lu tath io n e S -transferase, 4, 5 - B -esterase, 6 - m icro so m al m ix ed -fu n ctio n oxidases in h ib itio n , A - m eth y lp ara th io n , B - 0 -m eth y l-0 -p ara- n itro p h e n y l p h o sp h o ro th io a te (o r O -m ethyl-O -para-nitrophenyl p h o sp h a te), C - d im eth y lp h o s-
p h o ro th ic a te , (or d im ethyl p h o sp h a te acid), D - p a ra -n itro p h e n o l, E - m ethyl p a ra o x o n
d eco m positio n M P in the r a t’s organism is catalised by oxidases o f m ixed effect, hydrolases and g lu tath io n S -transferases.
G lu ta th io n e S-transferases (G ST - E. C. 2.5.1.18) are a m u ltig ene enzym e fam ily which perform protective functions against toxic effects o f xenobiotics [1, 7, 27], T h e study seems to be purposefull, because o f the lack o f univocal d a ta on th e subject o f possible processes w hich lead to a d am ag e o f cell genetic m aterial.
M A T E R IA L S A N D M E T H O D S
In this experim ent have been used m ale W istar ra ts w hich weigh from 2 5 0-30 0 g. M e th y lp a ra th io n (0 ,0 -d im e th y l-0 ,4 -n itro p h c n y lth io p h o sp h a te ), m inim um purity 98% (obtained from the In stitu te o f O rganic C h em istry
in W arsaw ), was adm inistered once into the tail vein 0,5 L D 50 (21 m g M P in olive oil per 1 kg o f body weight).
E xposure tim e to the insecticide was up to 150 m in utes. T h e prepared ra t tissues: liver, kidney, spleen, pancreas and brain, were kep t at the tem p eratu re o f -70°C . F ro m each o f these tissues was m ade h o m o g en ate so lu tio n in 0,25 M sucrose in 0,1 M p h o sp h ate sod iu m -p o tassiu m buffer (pH 7,4) on the to tal co n c en tratio n o f 20% (w/v).
E nzym e activity G S T was estim ated by the m eth o d o f co n ju g atin g G S H ( H a b i g ct al. [8]) with 1,2-dichloro-nitrobenzene (D C N B ) and 1 -chloro-2,4-dinitrobenzene (C D N B ). A ctivity was m easured in p o stm ito - c h o n d ria l, m ic ro so m a l an d cytosol fra c tio n s o b ta in e d by J a c o b y ’ s m eth o d [11] using 50 to 300 n g protein for C D N B and D C N B respec tively.
P rotein co n c en tratio n was estim ated by using L o w r y ’ s et al. m eth o d [13], G S T activity was expressed in specific activity units lU /ln m o l S -l- -c o n ju g a tio n form ed w ithin 1 m in u te in th e c o n d itio n s o f en z y m atic reactio n in term s o f m g o f protein. T h e results were analysed by S tu d e n t’s t-test an d confidential level appro x im atio n o f a = 0,05.
C hanges in G ST activity have expressed in % acco rd in g to the form ula:
S A p o is o n e d - SA control x ioO% SA control
w ere S A - Specific A ctivity.
R E S U L T S A N D D IS C U S IO N
In o u r w ork we have observed a statisticaly significant increase in the activity o f g lu tath io n e S -transferase tow ards D C N B and C D N B in all exam ined cell fractions in rats poisoned w ith M P in the liver, spleen, pancreas, b ra in and the kidney.
T h e degree o f the increase o f the activity o f cytosolic G ST was different in each o f the exam ined tissues. In the liver, the increase of cytosolic G S I to w ard s C D N B as a su b strate was 47% in the kidney, 53 /o in th e spleen, 19% in the pancreas, and 69% in the brain - Fig. 2. A s reg ard s the increase o f cytosolic G S T s to w ard s D C N B as a su b strate was 25,8 /o in the liver, 6,4% in the kidney, 34% in the pancreas, 18% in th e spleen, and 50% in the b ra in - F ig 3.
pancreas liver spleen kidney brain
F ig . 2. E ffect o f M P o n G S T activ ity in tissues o f th e W is ta r ra t. E nzym e activities w ere m ea su re d by th e co n ju g a tio n o f G S H to
C D N B
pancreas liver spleen kidney brain
F ig . 3. E ffect o f M P o n G S T activ ity in tissu es o f th e W is ta r r a t. E nzym e activities w ere m ea su re d b y th e c o n ju g a tio n o f G S H to
C ytosolic fractio n G ST s o f ra t liver belong to the best kn o w n enzym es o f this type. M an y isoenzym es are p resent there. T hey are protein s from 40 to 50 k D a , built o f tw o different o r identical su b u n its from 22 to 27,5 kD a. Till now, at least 10 subunits and 15 m olccullar form s o f this enzym e have been described in the ra t tissues.
T h e occurance o f isoenzym es o f overlapping su b strate specific qualities results from the existance o f a num ber o f d ouble co m b in atio n s o f sub un its.
V aried term inology is used to classify G STs. In o u r p aper a division in to 3 classes a, n, n has been ad o p ted according to M a n n e r v i c [14], Specific activity to w ard s the used substrates allow s, to a certain degree to d ra w conclusions th a t the classes a and fx, react m ainly w ith th e su b strate C D N B , while the isoenzymes 3-3 and 3 -4 o f the class ^ w ith D C N B [5], It is evident th a t the increase o f the activity in the liver concerned bo th , th e isoenzym es o f the class a and the class ¡i. Seven isoenzym es have been fo u n d in the pancreas [21]. T h e increase o f G ST activity in the p ancreas related also to isoenzym es o f the classes a and fi.
In the spleen, the increase o f G ST activity to w ard s C D N B was 50% an d to w ard s D C N B 18% . O n the basis o f division on S eph arose 2, tw o m ain form s o f isoenzym es 2 -2 (class a) and 7 -7 (class n) have been ch aracterized in the spleen, so the increase ol the activity related to the classes a an d n [23],
In the kidney, the increase o f cytosolic GST tow ards C D N B as a substrate w as 53% , and to w ard s D C N B 6,4% . A ccording to the investigations [22], in kidneys th ere are 8 isoenzym es G ST belonging to the classes a, fi an d n. O ur investigations show a p artic u la r increase of th e enzym es o f the a class. Enzym es o f the /i class also show a certain increase o f activity.
A n exceptionally high increase o f G ST activity has been observed in the cytosolic fractio n o f the r a t ’s brain. It was 69% in relatio n to C D N B an d 50% in relatio n to D C N B . It m ay be concluded th a t this relates to all classes o f cytosolic enzymes. A ccording to J o h n s o n ’ s et al. p ap e r [12], enzym es o f all cytosolic G ST classes a p p e ar in th e r a t ’s brain.
M icrosom al form o f G S T has a different stru ctu re th a n cytosolic form s. It has been exam ined in h u m an and ra t livers [15], It is built o f three identical subunits ab o u t 17,3 kD a. In o u r investigations, we have found in con siderable 10% increase o f m icrosom al G S I activity in th e p an creas an d liver, and a b o u t 18% increase in the spleen, while in the kidney, liver, an d b rain we have observed ab o u t 50% increase o f the G S T activity in re la tio n to to b o th exam ined substrates.
T h ere is very little literatu re d a ta concerning m icro so m al G ST o f the b ra in , so th ere is som e difficulty in th e in te rp re ta tio n o f th is result. P o stm ito ch o n d rial fraction contains b o th , the enzymes from m icro som al an d cytosolic fractions. T h e results for this fractio n seem to co nfirm ,
generally o u r observatio ns concerning the increase o f G S T activity in the exam ined fractions.
T h e ob tain ed d a ta clearly prove th a t the effect o f m c th y lp a ra th io n is an increase in the activity o f one o f the m ain enzym es o f the xen obiotic m etabo lism system.
O n the, -so far available, literature we h av e n ’t found d a ta related to the influence o f M P on G ST s o f all th e fraction s and tissues o f the rat.
Several new p apers on G S T s are given som e d a ta on the o f m icroso m al G S T in ra t tissues (also o f the brain) [16], the d istrib u tio n o f G S Il and G S T in m ito c h p n d ria and cytosol o f the nervous system [9, 17], th e G S H an d G ST s activity in the organs and blood o f ra ts follow ing ch ro n ic irra d ia tio n a t low doses [18], b u t none o f them uses M P as an in d u cto r o f G S T activity.
T h ere is also a p ap e r ab o u t the p u rp o se o f using cryopreserved tissue to study the m etabolism o f pesticides in fo o d -p ro d u cin g anim als an d rats [20] co ncluding th a t there is no q u alitativ e differances in th e results b u t a q u a n tity degrease appears.
T hese studies were supported by U niversity o f Ł ó dź g ra n t N r 555/255.
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W płynęło d o R ed ak cji D e p a rtm e n t o f M o le cu lar
F o lia bio ch im ica et biop h y sica G en etics
24.04.1998 U n iv e rsity o f Ł ó d ź
Z o fia W alter, M aria Gawrońska, C hristos Kargas, W anda M ajcherek
W P Ł Y W M E T Y L O P A R IA T IO N U N A IZ O E N Z Y M Y T R A N S F E R A Z Y S -G L U T A T IO N O W E J W R Ó Ż N Y C H T K A N K A C H S Z C Z U R A
W niniejszej p racy z b ad a n o wpływ insektycydu fo sfo ro o rg an iczn eg o - m e ty lo p a ra tio n u n a ak ty w n o ść tra n s fera z S -g lutationow ych w tk a n k a ch szczura ra sy W istar. Z b a d a n o frakcję p o stm ito c h o n d ria ln ą , cytozolową. i m ik ro so m aln ą w ą tro b y , n erk i, trzu stk i, śledziony i m ózgu. A k ty w n o ść o zn aczan o z d w o m a su bstratam i: C D N B i D C N B , co pozw oliło w yróżnić izoenzym y G S T . S tw ierd zo n o zm iany aktyw ności zależne od tk an k i i frakcji k o m ó rk o w ej.
