< ~ Prostaglandin 'levels and lysosornalenzyme activities in irradiated rats

P. J. TrochaG. N. Catravas

DEFENSE NUCLEAR AGENCY

ARMED FORCES RADIOBIOLOGY RESEARC 1rST1T UTEBETHESDA, MARYLAND 200)14

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RF N. CATRAVAS, D.Se. PAUL E. TYLER, M.D.(Thn irmtrt CAPT, MC, USNFli.cehmristry Depnrtment Director

Retaehwas tndueted aec-rding to the privnciples eruiiciated ini the-Guide for the Care and Vise of Laboratory Animals, 1" prepiared by thernstitute of tLb rttovy Awikal tsourcos, Nation~al Reqesreh CouneiL

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AFRRL-SR8 0- 28 O 0a-a,4. ILE 12d Subtitle) Pf aPERIOD CORE

-LROSTAGLANDIN IEVELS AND 4LYSOSOMALENZYME ACTIVITIES IN IRRADIATED RATS1

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Published in the International Journal of Radiation Biology 38: 503-511, 1980.

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Whole-body irradiation of rats results in the release of hydrolases from lysosomes,an increase in lysosomal enzyme activities, and changes in the prostaglandin levels inspleen and liver tissues. A transient increase In the concentration of prostaglandins Eand F and leakage of lysosomal hydrolases occurred in both spleen and liver tissues 3-6hours after the animals were irradiated. Maximal values for hydrolase activities,prostaglandin E and F content, and release of lysosomal enzymes were found 4 days

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20. ABSTRACT (continued)

postirradiation in rat spleens whereas in the liver only slight Increases were observedat this time period for prostaglandin F levels. On day 7 there was a final rise in thespleen's prostaglandin E and F concentrations and leakage of hydrolases from thelysosomes before returning to near normal values on day 11. The prostaglandin Fconcentration in liver was also slightly elevated on the 7th day after irradiation andthen decreased to control levels.

i oation - - ' - '

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Prostaglandin levels and lysosomal enzyme activities inirradiated rats-1

1). J.TI'R(CIIA and G. N. CATIAVASIiochemnistry D~epartment, Ai niied Forces Radiobiology Rtesea rch Institute,llethesda, Mlaryland 200)14, U.S.A.

(Received 12 Decemtber 1979; accepted /0 MaY 1980i)

W~hole-body irradiation of rats results in the release of hydrolases fromlvsosomes, an incre-ase in lvsosonial enzymie activities, and changes in theprostaglandin levecls in spleen and live#.r tissues. A transient increase in thlecono-entration of prostaglandins E and 1: and leakage of lysosomal hy'drolasesoccurred in both spleen and liver tissues 3 -6 hours after the anlimals wereirradiated. 'Maximal values for hydrolase activities, prostaglandin E and Fcon tent, and release oflIvsosomalI enzymes were found 4 days posti rraidiation i n ratspleens whereas in the liver only slight increases were observed at this time periodfor prostaglandin F levels. On day 7 there was a final rise in the spleen'sprostaglandin E' and F concentrations and leakage of hydrolases from thelvsosornes before returning to near normral values on day I I. The prostaglandin Fconcentration in liver wvas also slightly elevated on the 7th day after irradiationand then decreased to control levels.

1. IntroductionRecent evidence suggests that ionizing radiation affects the prostaglandin levels

in animal tissues (Eisen and Walker 1976, P~usescu, Chirvasie, Teodosiu and Plun1976, Pryanishnikova, Zhulanova and Romantsev 1978). These changes inl pros-taglandin levels have been found to increase within several hours after exposure ofthe animal and to remain elevated for days, especially in the radiosensitive spleen andthymus tissues (Eisen and WValker 1976).

Additional evidence has been accumulating to show that synthesis and leakage ofenzymes from lvsosomes are also affected by radiation (Watkins 1975). Lysosomalhvdrolases and proteinases have been found to increase and reach abnormally highlevels in many mammalian tissues several days after radiation treatment (Snyder1977).

Previous studies (Eisen and Walker 1976, P~usescu el a/. 1976, Pryanishnikova elal. 1978, Watkins 1975, Snyder 1977) suggest that both prostaglandins andlysosomes are involved with a mechanism that controls the inflammatory process intissues injured by ionizing radiation. Other investigators (Ignarro 1975) have tried toimplicate prostaglandins and lvsosomes in the tissue injury process. But muchconsiderable controversv continues as to whether prostaglandins are actuallyinvolved wvith the stability of lvsosomal membranes, since these studies used in vitroenvironments (Ignarro 1975). Therefore, this investigation was perform-ed using an

t Supported by Armed Forces Radiobiology Research Institute, Defense NuclearAgency, under Research WVork Unit MJ 60413.'l'he views presented in this piipcrare those 4fthe authors. No endor'ement of the lDefeibe Nuclear .\gcnc has been given or should beinferred.

Coyih 114511 I'S. Ctnn

504 P. J. Irocha and 6. N. ('atravas

ill viv' system to determine if prostaglandins and Ivsosomes are influenced byionizing radiation. If they are interrelated, then the ef'ect of prostatlandins on theintegrity of the lysosomal membrane can be deteriined.

2. Materials and methods2. 1. ( 'hemicals

llhcnolphthalcin fl-glucuronide, 4-methylarninophenol, glcero-2-phosphate.and silicic acid (Sil 13-200) were obtained from Sigma Chemical Co., St Louis, MO.Amersham (Arlington Heights, IL) suppplied [5,6(n)-311] prostaglandin E, and[5,6,8,11,12,14,15(n)-S3l]prostaglandin l2, for tracer studies. I I prostaglandin Eand !F2, RIA kits were purchased from Clinical Assays, Inc., Cambridge, MA.

2.2. AnimalsA total of 750 male Sprague-Dawley rats, 7 5-15 0 g, were used throughout the

investigation. They were divided into three groups of 250 each, in which two rats percage were kept in a room maintained on a 12-hour light (0600-1800) and 12-hourdark (1800-0600) cycle. All the animalswere given a Wayne Lab Blox diet and waterad libitum.

2.3. Irradiation of animalsFrom each group, 190 rats were placed in Plexiglas restrainers and exposed

bilaterally to 1000 rad of 6 Co radiation at a dose rate of 50( rad/min. The remaining60 rats were kept as sham controls. At designated time intervals after irradiation, 12-15 exposed and 4 sham-irradiated control rats were sacrificed by exsanguinationunder ether anaesthesia.

'2.4. Preparation of tissue homogenates and enzyme assaysSpleens and livers were excised from rats and frozen in liquid nitrogen except for

0-05-0-1 g of tissues from each organ. The portions of the organs that were quicklyfrozen in liquid nitrogen were later used for assaying prostaglandin concentrations.The fresh sections of spleen or liver tissue were gently homogenized in 2 ml of 0"2 MKCI1 using a Dounce homogenizer. One half of each homogenate was centrifuged at12 000 g in a refrigerated centrifuge for 10 min in order to obtain a supernatant free oflvsosomes. The remaining half was frozen and thawed twice. The lysosome-freesupernatants and thawed whole homogenates were then assayed foracid phosphataseand B-glucuronidase activity (Barrett and Heath 1977).

2.5. Isolation and purification of prostaglandinsTissues from rats frozen in liquid nitrogen were weighed, homogenized, and the

prostaglandins extracted no later than 1 hour after the animals were sacrificed using amodification of the method employed by Jouvenaz, Nugteren, Beerthuis, and VanDorp (1970) as shown in figure 1. Each liver sample, weighing 3-4g was quicklyhomogenized in ice-cold ethanol containing 3 H labelled prostaglandin E or F tracerfor 30 s with a Polytron (Brinkmann Instruments, Inc., Westbury, NY). However, itwas necessary to pool four or five spleens to obtain an adequate amount of spleentissue (0"5-3 g) for each prostaglandin determination. This method results in a 30-55per cent recovery ofprostaglandins found in rat tissue. Corrections for prostaglandinlosses in each sample were made when calculating their concentrations.

El.Tects of radiiation an NIsisones aid prostaglau/ins 505

TISSUE 10.6 4.0 . FROZEN IN LIQUID NITROGEN HOMOGENIZED IN 30 40rolEtOH ICONTAINING

3H PCI AND CENTRIFUGED

- PRECIPITATE DISCARDED

EiOH EVAPORATED UNDER VACUUM AT 4SC. RESIDUE DISSOLVED IN 0.05 MTRIS. pH 7.5 (1.5 m AND PARTITIONED WITH EtAc (2.0 ml) TWICE.

SEtAc DISCARDED

AGUEOUS PHASE MADE pH 4.5 WITH 0. M CITRATE. pH 4.0 (0. IM) AND PAR-TITIONED WITH EIAc (3.0 odl TWICE.

, AOUEOUS DISCARDED

POOLED EtA. WASHED WITH I40 (0.2 riW AND EVAPORATED UNDER VARIJUMAT 371C. RESIDUE DISSOLVEU IN 2.A % M.OH IN CHNC3 (0.2 ad) AND FRACTION-ATED ON 0.6 n 9 cm SILICIC ACID COLUMN:

FRACTION I IDISCARDEDI 2 nA OF CHC 3 4 2 Ml OF 2. % M H IN CHCI3FRACTION 2 (PG A) 8 mn OF 2.5 % M.OH IN CHCI3FRACTION 3 IPG E) 10 mI OF 5.0 % M@OH in CHC13FRACTION 4 IP F) )d OF 20 FMOH IN CO 3

Figure 1. Scheme for isolating prostaglandins.

A comparison of our procedure (figure 1) with others in which indomethacin(Vane 1971), or acid (Hensby 1977), was included in the homogenization media was

made in order to determine if any artifactual prostaglandin synthesis occurredduring its isolation. It was found that after correcting for losses, the concentration ofprostaglandins isolated from rat tissues was essentially the same when using eitherethanol or homogenation media which contained indomethacin or acid. In anadditional study rats injected with indomethacin (10mg/kg wt. of animal) 2 hoursprior to their sacrifice had prostaglandin concentrations the same as the untreated

controls when analysed by the method shown in figure 1. Therefore, employment ofour technique allows the determination of the in vivo levels of prostaglandin in ratspleen and liver tissues.

2.6. Assays of prostaglandinsAnalysis of purified prostaglandins E and F was performed by immunoassay

(Jaffe and Behrman 1974). 'he binding characteristics as well as the degree ofprostaglandin cross reactivity fir the commercially available antisera that was usedin this study have been previously detemined by Jaffe and Behrman (1974) andLevine, Gutierrez Cernosek and Van Vunakis (1971).

2.7. StatisticsStatistical analyses were based on the mean that was calculated from the average

values of three separate experiments. Therefore, a total of 12 control and 36-45experimental animals were used per time interval for calculating the appropriatemeans and standard errors. Differences between experimental and correspondingcontrol values for the same time interval were considered significant if Student'sunpaired t test gave a probability (p) of less than 0"05. The experiments weremonitored for a period of 11 days.

3. Results3.1. Liver lysosomal enzyme activities

fi-glucuronidase activity was measured by the release of phenolphthalein fromthe substrate phenolphthalein-fi-glucuronide. As shown in figure 2, the 11-glucuronidase activity of the enzyme was not significantly different in the irradiatedrats as compared to the corresponding nonirradiated controls, although a slight but

506 1'. J. "rocha and (..V. (atraiI.s

400-

3 100

2W

0 4 --0 0.25 0 2 3 4 5 a to 10

TIME (dos)

Figure 2. ltfect of ('Co radiation on liver fl-glticLrtmnidase activity in rats.- tnfractionated liver experiment:d - .... unfractionated liver control; A..- 12 (X) g liver supernatant experimentail fraction; -.-/- . 12 00 g liver super-

natanlt control fraction. Mleans + S.E. S.I". indicated by vertical lines. p > 0-05 (towardcorresponding control) for all time intervals. n=9.

insignificant increase was observed 3-8 days after exposure. fl-glucuronidaseactivities in the lysosome-free 12 000 g supernatants from the experimental and the

control liver-tissue homogenates also showed no changes (figure 2) except for a smallnonsignificant elevation at 6 hours postirradiation (p=0 19). No significant changes

in acid phosphatase levels in the liver samples were observed following ionizingradiation (data not shown).

3.2. Sp!een 1.'sosomal activitiesAn increase in the lysosomal fJ-glucuronidase activity was observed in the spleen

following irradiation. Within 3-6 hours after exposure, its activity began to rise,

reaching a maximal level in 3-4 days, which was four times greater than the controllevels (figure 3). After day 4 the enzyme gradually decreased until day -10, when it

no

20

0 025 0.50 1 2 3 4 a a r a 9 T0 11

TIME (d1l

Figure 3. Effect of 6 0Co gamma radiation on spleen /l-glucuronidase activity in rats.-E- =unfractionated spleen experimental, - -- --- unfractionated spleencontrol; - @-= 12000g spleen supernatant experimental fraction; -- 0--= 12 000 g spleen supernatant control fraction. Means + S.E. p

" l.'eels 4 radithim, ni /vssmes and pros, 507di7

rcturned to a normal physiological level. Similar iji, teases were observed with acidIlhosphatase activity.

Figure 3 also shows that the /f-glucuronidase activity level in the lvsosome-freehomogenates (12 000g supernatants) rose insignificantly within 3-6 hours (3 hours,p = 026; 6 hours, p = 008) after irradiation of the animals and continued to increaseuntil day 4. The activity then slowly decreased to near control levels. Similarpatterns for acid phosphatase activities were found.

3.3. Percentage o soluble lysosomal enzyme activitiesFigure 4 illustrates the percentage (if soluble f-glucuronidase (!2 (X0 g super-

natant) found in the unfractionated homogenates. Within 3-6 hours after irradiationof the animal, the percentages of soluble enzymes released from the liver and spleenlysosomes were significantly elevated 15- to I 8-fold (6 hours, p = 0"05; 3 and 6 hoursspleen, p

50S P. J. "rw/h, and G, N. 'alralves

A'

fT

77

' T

to 1 ' 0 6 1 8 o I

Fig'ure 5., -fc of "'Co gamma radiation on the prostagl indins iii nat liver. PGE values, in (A); P( concentrations show..n in (B).......=experimental;

---=- ,,trol. N[eans+S.E. p< 5 (toward corresponding liver control in (A) for0 PG E sampes. p

/'flects j/ radiahm fm,, ./sows and prealqgtaespins 509

A

1000.

,,--4 ---= -. . --. . -7tB

It

1000

51000.

l00

0 02S 05 1 2 3 4 b * " S 0 O 0 11

DAYS

Fiti.ure 6. EIffect of '"Co gamma radiation oni the prostaglandins in spleen. PGE valuesillustrated in (A); P(F concentrations shoW1n in (B).... -experimental;----- =control. leans + S.E. p

lii 1'. 1. 'I'rv1mchi' till(/ G,. N.( ima

4.2. Pait'irs r 'xponsil .fin- r-4e'/ase of I'svsmnlplal 'lv'sr anid iiicremed x'gI pr, iiglsdi,,

Thei decrease in size and weight (if the spleenl that occurs %\ithin hours aftereXp)osure' to "'Co gammna raidiation is minly dlue to pvknotic changes and dleath ofthe highly radiosensitive lvmphoid cells. Phagocvtosis of these in~iured cells byniacrophigcs and reticulum cells reaches maximal activity 3 hours after exposure inthe spleen (Jordan 1967). Thel phaigocvtic pr. bCess Is Complete I day after exposure.At this time, the spleen has last 7n) -80 per cent of it, weight. This process, as well asrelease oft enzymes from the disrupted lysosomes, could be responsible for the abruptrise of prostaglandins 3 houis after irradiation, since I liggs, McCall, and Youlten(1975) have fouind that white blood cells engaged in phagocytosis release pros-taglandins, which then attract more phagocytes. I lowever, the highest prostaglandinlevels and greatest release of lvsosomal enzymes occurred several days after radiationexposure. Bly this time, the phagocvtic activities have ceased and the spleen ha.4started to regenerate (Jordan 1967). Consequently, there must be other agents thatart- responsible for the secondary increases in prostaglandinl levels and release/enzymes from the lysosomes. E~isen and Walker (1976) have observed that theelevation in prostaglandin levels which occurs several days after irradiation might becaused by a reduction in the rate of prostaglandin degradation. It is indeed possiblethat several enzymes, such as 15 dehvdrogenase or 13,14 reductase wvhich areassociated wvith inactivation of prostaglandins might be altered after irradiation.

Radiation damage could also affect other factor-, related to prostaglandinsynthesis. Recent investigations have found that 2-4 and 7-10 days after irradiationthere are fluctuations in cyclic nucleotide levels ('lrocha and Catravas 1980),disruption of tight junctions in the ilea of tihe intestine (P~orvaznik 1979),inflammation of numerous interstitial tissues (Walker, personal communication),high levc-ls of endotoxins and/1or bacteria in spleen and liver (Walker, Ledney andGalley 1975). The above reports indicate that these cell components may be involvedin the observed seconJary increase in prostaglandin concentrations and release oflvsosomal enzymes after exposure of the animal to radiation. In fact, the bacteria andendotoxins could be the main factors which perturb the cell, causing leakage oflvsosomal enz mes and synthesis of prostaglandins by disrupting the cellularmembrane.I

Therefore, the initial and secondary increases in prostaglandin levels and releaseof lvsosomal enzymes could be attributed to membrane disruption in the followingwvays: (a) breakage of mammalian cells by direct interaction of cellular membraneswith gamma rays, or from the free water radicals produced by 60Co exposure; (b)secondary rupture of cellular membranes due to their exposure to invadingendotoxins and bacteria. Disruption of the membranes by these factors could affectenzymes and lipids which are associated with the synthesis and increase in theconcentration of prostaglandins following radiation exposure. Formation of theseprostaglandins would then destabilize lysosomal membranes, resulting in thedispersal of lysosomal enzymes throughout the cell.

L'irradiation totale die rats conduit ai une perte des bydrolasems des lysosomes, une,augmrentation tie Paictivit des enzymes des% lysosomes, et des ch-.ngements des niveaux desprostaiglandines dlans la rate et le foie. line 16vation temnporaire die fit concentration desprostaglandines E et F et une fuitedes hydrolases des lysosomeq ont &ti prod uites dans la rateaussi bien que dans le roic entre 3 et 6 heures apr~s l'irradiation des anitnaux. Lees valcursmaiximumn de l'activit:6 des hydrolases, die la teneur en prostaglandines ~ect Fet ce la perte des

Iells ,f radia tin, oil li'Sssnes and Prostaglan~dins 5 11

cilI lies ties ISuosollics oquit tC' I.'uusst troutivcs tiass Ia nate 4joursapr4&s irraudI ita,,. I'drt(Jntre,d. its le foic. scu lei nent di lvtitcs eleatanais di fivetu t la priastaidandi ti F (ot etc trouv csiicc naumilent 111. 11 cut title a ugmentation finale des concefltrltionseni prostagIandioes E ct I- etdic Ia perte ties Iivdroiases ties Ivsosomecs dans la rate ato cours dui 7ilnw jour apris irradiation.suivies d'un retour it des valcurs, a puti prini nonnales dec I Ierne jour. L a concentration enprostaglandine F darks le foje tait aussi ltg~rcmnrt decv~c au 7Z-me jour apr~s tjuoi cite adiminuell Ili nivcau des t~nioinls.

Gaizkiperh s trahiung %-il Ratten befreit die I iydrolasen der Ly.sosonie, eriiilbt die:Aktivit~it %-on Enzx-nen dier I AsIosofe nd verindert dais P~rostaglandin (*;eicbgewicht in derMdxil und dier Lch er. Man bcmnerkt cine voriibergethenide Zunaine in der Konzentration derI'rostag.indin E. und F und cine I o(kering der I ivdrolasen der Lysosornen in der Milx undder Leber. 3 (6 Stunderi nach Brestrahiung der TIiere. IDer maximale Wert-fulr die Aktivitiittier I tvdroiasen, der EProstaglandine F und F, und der Enzy me der Lysosome w~urde 4 T[agenach Bestrahiung in der Nlilz dier Ratter, gesehen; w~ibrend nur eine kleine Steigerung desP~rostaglandin F zur selben Zeit in der Leber zu sehen war. Die Ietzte Steigerung derKonz'ntramtion von Pro~taglaridin F tntd F tint) tir I lydrolasen der Lysosome der Miiz waraml / .''ag~e. Norniale Werte ersebeinen am I I.Tage nach der Bestrahiung. Die Konzentrationdes P'rostagzlandin F in der I en war etwas erht am 7.1Tage nach der Ilestrahiung.

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104.RENi . .. ,.DARDEN, J. I I., and PARtKEII, J. L., 197 1, Lab. Invest., 25, 230.SNYDJI', S. Li., 1977, Radial. Res., 69, 306.TR(iA. P., and CATHAVAV;. G. N., Radial. Res., (in press).VANE, J. R., 1971, Vature, Vetc Biol., 231, 232.WAI.Ki at, R. L, I I)NEY, G. D., and GALLI.Y, C. BI., 1975, Radial. Res., 62, 242.

1~l s ). K.. 1975., Ltvsosonmes in Biology, and Pathology, Vol. 4, edited by J. T. D~ingle andIt. T. [)eain (New York: American E Isevicr) p. 147.