Lysosomal injury and hepatic necrosis

J.;SI’I:I1IMI~:NT.\I,

.,X1)

MO,,1~:~‘I~,>.\,1

Lysosomal Effects

of Triton

I’\THOI,O(:Y

Injury

14, :%jo-361

and

Hepatic

on

WR-1339

(l(3;l)

Liver

Necrosis Cells

in the

Rat’

It has been demonstrated in a lnwious papw t,h:tt Triton WR-1339 administration aggravates carbon tetrachlori(lc (CClr) -induced liver necrosis in the rat (Iturriagn, Posslaki, and Rubin, 1969). It w Ls suggested that this effect could bc clue to the lysosomal injury producccl by the dctergcnt. I?& vi,Uo administration of t,lic nonionic detergent Triton ‘ll?R-1339 has several effects besides labilization of lyrosomal membranw. Thr best known of them is the production of hylwrlit)ennia, l)rol)nbly due to a modification of circulating lipoproteins iFriedman and Bycrs. 1957), Hcmolyais has also been observed (Scanu et nl., 1961‘I . and thv t)rotluctioll of mitochontlrial ~dnmagewhen Triton was added to animal cell cultures was rccc,ntly reported (Zimmerman et al.: 1969). Therefore there is a possibility tllnt. the action of Triton r&l&s alterations of many lipoprotein membranes. The purpose of this paper is to communicate further investigation on this problem, which has been oriented in two directions: (a1 To exclude the possibility that the est,ensivc necrosis ~~rotluwtl by the combination of Triton M’R-1339 and CC& roul(l be the result of a peculiar intrraction between these two compounds, experiments wing thioacctamidc instead of CCl, wrc performed. The magnitude of necrosis was waluatctl by light. mirrotico~)y. (b) Effccta of Triton on cell st,ructure were studictl by dctcrniinntion of the activities of enzymes located in different cellular compartments. ’ This work IKE supported 1)~ Iiniversidad de Chile. Invcstignci6n Cientificn, Proyerto 65-42 (3.107.4401). 350

Fncultntl

de Medicinn,

Comisibn

de

LYSOSOMAL

INJURY

AND

HEPATIC

NECROSIS

351

Sorbitol dehydrogenase, an enzyme localized mainly in the soluble fraction of liver cells, was determined in serum and its elevation was considered as an index of cell membrane damage (Asada and Galambos, 1963). Succinic and glutamic dehydrogenases, two enzymes localized in the membrane and matrix of the mitochondria, respectively, were determined in the soluble fraction and used as markers for mitoehondrial membrane alteration. Acid phosphat,ase distribution in soluble and sedimented fraction was considered to est,imate lysosomal membrane damage. Electron microscopic studies were also performed and corrclat’ed with biochemical results. MATERIALS

AND

METHODS

Dornyu white malt rat,s weighing 140-220 gm were injected into a tail vein with 0.65 ml/100 gm body wt of Triton WR-1339 (Ruger Chemical Co., Inc.) in a saline solution containing 200 mg/ml. Animals were sacrificed serially after the injection in periods ranging from 90 minutes to 24 hours. Control group was similarly injected with saline solution. When thioacetamide was used, it was injected subcutaneously at, a dose of 0.5 ml/100 gm body wt in a saline solution containing 20 mg/ml. Animals were fasted for 12 hours bcforc sacrifice. Iinder light ether anesthesia, a blood sample was obtained from the aorta for serum sorbitol dehydrogenase determination. After complete exsanguination, the liver was quickly removed, weighed, placed in ice, and divided in scvcral pieces for chemical, electron microscopy, and histologic st.udies. Histologic

studies

Blocks of liver were fixed in 15% neutral buffered formalin; paraffin sections were prepared and stained with hematoxylin-eosin, Mallory’s chromotrope aniline blue, and Maximow stain (Maximow, 1909), Electron

microscopy

In four rats from groups sacrificed at 1.5, 6, 12, and 24 hours after Triton administration, samples obtained from the left lateral lobe were fixed in 1% osmic acid using phosphate buffer at 0°C. After alcohol dehydration t.hey were embedded in Epon 812, according to Luft (19611. One-micron thick sections were prepared to select desired areas, which always included rentral and periportal zones. Ultrathin sections were cut with a Porter-Blum MT 2 ultramicrotome, using glass knives. They were stained with uranyl acetate-lead citrate and examined in a SiemensI Elmiskop electron microscope. Chemical

studies

Acid phosphatase detewnimtions. One gram of liver was homogenized in 9.0 ml of 0.25 dl sucrose at O”C, using a glass homogenizer with a Teflon pestle; 3.0 ml of this 10% homogenate was rehomogenized in 7.0 ml 0.25 Jf sucrose and centrifuged at 0°C for 30 minutes at 14,000g in an International PR 2 refrigerated

352

HERNkX

ITURRIAGh

E2’ AL.

centrifuge with multispced :tttjachmcnt,. The supernate const,ituted the soluble fract.ion. To dctcrrnine total activity, 3.0 ml of the 10% homogenate was rehomogenized with 7.0 ml of 0.25 111sucrwx-1’4 Triton S-100. Acid phosphatasc activity was determined in 1.0 ml of each fract)iun, using glyccropl~ospllwte as substrate. Rcleased phosphorus was estimated accorcling to Fisk anal SubbaRow ( 1925) Activity was exlnwwd as mirromoles P released in 10 minutes per total liver per 100 gm body wt. Dehydrogenase dctc~rminations. C)ne gram of liwi- was similarly l~oniogenizcd in 10.0 ml of 0.25 dl sucroscl-I .O mX JXI>Th-6.0 m?iI Trk buffer ipH 7.2-7.4). The homogenate was centrifuged at BOOgfor 10 miuutes at. 0°C to eliminate nuclei and intact cells. 011~1 rnillilitcr of the supernate was rcsuspcnded in 4.0 ml of 0.25 M s:ucro.~e-F:l)TA~‘I‘ris nutl cc~ntxifugc~tl for 30 minut,es at O”,C and 14,000g. The supernate constituted thr soluble fraction. Tllc~ sediment w-as rcsuspendcd in Tj.0 ml of 0.25 X sucrose-EDTA-Tris and rchomogcnixed in an UltraTurrax homogenizer for 2 minutw at 0°C. Glutamic clehydrogcnwsc (bX 1.4.1.2, r,-glutamate: NAI) ositloreductxsel was determined according to Strcckcr ( 1953) , using glutamate as suMrate. NADH accumulat,iorl war mcasurcd with a spcctrophotometer Beckman D.1;. with Metrolim recortlcr nttachmcwt. Succiiiic tleliyclrog~w~sc ( 1%’ I .3.99.1, suwinatc~ or;itlorctluctasc~) was asbaycd by the method of Slater ail11 Holiner ( 1952) mcwsuring the rctluction rate of &Fe (CN) ,;, IGlzyme assays wcrc lwrformetl xt 20°C’. Activities were ~q)rcwccl in International I-nits (11-J at 20°C. Total activity was obtained by addition of soluble and sedimented fractions and rcfcrrcntl to t’otal liwr,./lOO gni hotly wt. Sorbitol dehydrogenasc. This was assayed in 0.3 ml of serum. using sorbitol as sub&rate and measuring the fructose formed by the colorim&ric mcthocl of Roe et nl. (1949). Before reading, all samples were treated with 5.0 ml of cliloroforni to eliminate turl)itlity, duca to Triton-induccvl hyperlipmllia.

Livers of Triton-treated animals TVC~W normal csccpt for moclcratc~Kupffvr cell hyperplasia and light sinusoitlal dilatation (Fig. I 1. In animals rcwiviug only thioacetamidt~ and sacrificecl after 24 hours, it mild central necrosis was always present, without affecting more thau l;iCC of the total p:~wnchym:~ 1Fig. 2). In necrotic areas a niotlc~rntt~to swtw inflammatory reaction dad ol~crvcd, with :I mixed infiltrate romposccl of ~~olyi~ior~~lionut~le:lr and mononuclear cells. When Triton and thioawtamiclc wrc administcretl simultnnc~ousl~, the lirer of animals sacrificed at, 24 hours was severely damaged. Necrotic areas extended beyond the central zones confluent with similar areas of adjacent, lobules. In average more than 4Of! of the total parenchyma was affected. The general aspect was that of a sulxnassire necrosis (Fig. 3). In the center of necrotic zones marked inflammation with ~,olpnlorpllo~lurlear leukocytes was observed.

Z;lu. 1. Section of liver from ran trcxtc’d with Triton kY;K-1339 and swrificcd ;iftrr t.hc injection. H & E stain. Xl00 original magnificntion.

FIG. 2. Section of liver from rat sacrificed 24 hours after injection mow stain. Xl00 original magnificat.ion.

at 24 hours

of thioncetamide.

Maxi

FIG. 3. Section of liver from rat sncrificed thioacetamide. Masimow stain. Xl00 original

34 hours after magnification.

injection

of Triton

WR-1330

and

Electron m~‘c~oscopy Livers of control animalh lnesentccl the usual morphologic findings (Fig. 4). In animals receiving Triton, the most prominent feature xts the presence of single membrane vacuoles, of diffcrcnt size, usually located near the biliary pole and partially filled with a dense osmiophylic material (Figs. 5 and 6). These vacuoles were found at all periods stuclied, being more numerous at 24 hours. Bile canaliculi were usually dilakd with spar&p or disappearance of microvilli. The rest of the cell organclles were normal. Sinusoidal membrane did not show detectable changes (Fig. 7j. Biochemical studies Acid phosphatase. Total activity did not change significantly after Triton administration except for a decrease observed at 90 minutes. Activity in the soluble fraction, expressed as percentage of total activity, increased significantly at 90 minutes after the injection and remain elevstcd during the whole 24-hour period, with higher values at 12 hours (Table I and Fig. 8). Dehydrogcnascs. Succinic dehydrogennsc did not show differences at any period, compared to controls (Table II). Glutamic dehydrogenasc act,ivity in the soluble fraction ~xhibitcd a moderate incrcasc at 24 hours (.05 > p > .02). Total sct.ivity xliow~tl some \-ariat.ions at, difftr~nt lwriorle, Iwing dcrrcnscd at 90 minutes (p < .05) ard t~lwntctl at’ 18 hours (p < ,005) (Table II). Sorhitol clehydrogcnnsc. The serum activit’y of this enzyme remained at normal

LYSOSOMBL

Fro.

4.

FIG. 5. jection.

Electron

micrograph

Electron micrograph Arrows indicate lysosomal

INJURY

of liver

AND

from

control

HEPATIC

animal.

of liver from Triton-treated vacuoles. X18,000.

355

NECROSIS

X10,500.

rat

sacrificed

24 hours

after

in-

FIG. 6. Electron tion. ArroLvvs indiwtc

microgrnlh lysosomnl

of liver vncuolcs.

FIG. ject.ion.

microgruph

of liver

7. Electron X20.000.

from Triton-1 X20.000.

from

rr:~icti

Triton-trwted

I’:II s:idiwil

rat

xwrifiwd

24 1101irs :ifti*r

12 tlours

nflcr

irijt+

in-

LYSOSOMAL

INJURY

ilND TABLE

I~IVER

PCID

PIICJSI,H.~T.\SE

AFTER

TRITON

HEPATIC I

AI>MINISTR.\TION

____~__

Group

Number

Control 90 Minutes 3 Hours ti Hours 12 Hours 18 Hours 24 Hours

40

SOLUBLE

of rats 11 8 7

9 16 7

12

ACID

PHOSPHATASE

0

acrd phosphata? f% m soluble fractlon.1

m

sorbitd dehydrogenase of serum1

30

(ME.\N

.\NI)

ST.\ND,IRD

ERROR)

.~~.

a rmoles P/total liver/100 gm body b p < ,001 compared to control. c p < .05 compared to control. LIVER

357

NECROSIS

Total

activity”

Percentage

in soluble fraction ____-

293.9 208.1 301.9

* f f

9.ti 12.5* 3-1.0

9.G * 0.8 17.2 f 1.7* 16.9 & 1.8*

355.8

zt

32.5

309.G 313.9 331.1

* + f

25.9 20.2 13.oc

22.9 24.7 24.1

f 1.5* f 2.9* r!L 2.7b

23.fi

+

2.3h

wt.

AND SERUM ADMINKTRATION

SORBITOL CEHYDROGENASE fM.an md SE. I

AFTER

TRITON

of actrvity

II U/ml

20

IO

contrd FIG. 8. phosphat,ase phosphatase fraction.

I

90 min.

3 hrs.

6 hrs.

Activities of srrum sorbitol in liver homogenates. after is expressed as percentage

12 hrs.

I8 hrs.

24

hrs

dehydrogenase (mIU/ml serum) and soluble acid different periods of Triton injection. Soluble acid of total activity which was found in the soluble

levels during the first 3 hours after Triton administration. From 6 hours it increased significantly, reaching higher levels at 12 hours (Table III and Fig. 8). DISCUSSION Results herein presented confirm previous observations t’hat Triton WR-1339 administration aggravates liver necrosis induced by some chemical hepatotoxins in the rat (Iturriaga, Posalaki, and Rubin, 1969). Use of thioacetamide produced similar effects to those observed in CC14 intoxication. Necrosis aggravation obtained by using two chemical compounds may be explained in several ways. One of them is Lhat it represents a simple addition of

3.58

HERXdN

Succinic

Sum-

Gr0llp

ber of rats

Control 90 Minutes 3 Hours 12 Hours 18 Hours 24 Hours

7 8 8 8 7

8

dehytlrogenase

Total activity” 78.7 liY.6 90 .3 88.X 89.6 83.0

f f f i f f

5.4 3.ri 6 .O 2.8 l.!) 3.1

50.8 51.1 52.1 52.0 50.8 ,53.:< gnl body

T.4RLE ~ORHITOL

Glutamic

Percentage in soluble fraction

u International Units/total liver/100 b p < .05 compared to control. c p < ,005 compared to control.

Smr.n4

ET SL.

ITURRIAGA

J)EHYDIWGI.XASE (MEAN

Group Control 90 Minutes 3 Hours 6 Hours 12 Hours 18 Hours 24 Hollrs

.\XD

+ f + i i It

1.:j 1.1 1.0 0.X 0.11 1.3

Percentage in soluble fraction

.58,(i

f

6.5

:tn.ci 50.1 73.1

zt i f

2.2”

90.4

f

i.lir

60.0

i

3.x

7.7

9.1

28.9 27.9 23.5 28.5 30.X 39.0

f f

3.1 2.9

f

3.7

f

2.7

+ f-

3.1 x.2*

III

QT.AND.\III)

of rats 13 1% 8 i 5 7 7

Total activity”

wt

.%FTF:R

Number

dehydrogenase

'~I~ITON

ADMINIhTlt.\TIO)N

ERROR)

Activity

(mIU/ml 3.1 1.8 X.0 18.8 35.2 31.0 33.3

ziz xt z?z -f f f *

serum) 0.81 0.5” 3.v 4.7b 12.-Lh 3.4* 8.V

a Nonsignificant, compared to control. I p < .OOl compared to control.

effect.s. This possibilit’y can be ruled out since Triton, by itself, does not produce detectable necrosis. An alternative to be considered is t’he csistence of some kind of interaction bettwcn the two rompounds. It has been reported, for example, that the CC14-inducrd hepatic lesion is aggravated by phenobarbital administrstion. It is believed that toxic effects of (‘Cl, are due to an int,crmediate product of its metabolism, which occurs in tlic microsomal drug-n~etabolizing system of liver cells. Phenobarbital would enhance 021, toxicity through its act,ions on t.he microsomal system iGarner and McLean, 1969’1. Triton did not product changes in the endoplasmic reticulum, and the aggravating effects on liver necrosis are not specific for CC14 since they were reproduced with the use of thioacetamide. Although mechanisms cqlaining thioacctamide-intluwd liver necrosis arc not fully understood (L\IcI,ean et al., 1965) a specific interaction lwtwcen Trit’on and the two hrpatotosins used stems, at present, unlikely. To investigate a possible damaging &ect of the detergent on cell membranes, several enzymes were assayed, both in total homogenates and in the ccl1 com-

LYSOSOMAL

INJURY

ilND

HEPATIC

NECROSIS

359

partment where normally they are not present. Such a method could be questioned on the basis that there may exist membrane alterations without producing abnormal permeability to enzyme molecules. Although this objection is valid, the opposite is also true in t’he sense that minor alterations, like disturbances in cation transport for example, can hardly he correlated with as complex a phenomenon as cell death (Judah, 1969). It is well known that cells can survive even severe biochemical changes, for example, the acute ATP deficiency of ethionine intoxication (Farber et al., 1964). Wit’11 the methods here employed it may be concluded that Triton administration produces an early lysosomal injury, reflected both in the increase of soluble acid phosphatase and in the ultrastructural observations. Although no cytochemical studies were performed, the lysosomal nature of the vacuoles observed has been previously demonstrated (Wattiaux et al., 1963) and is further supported by t’he common presence of organelle debris inside them (Fig. 6). During the first hours after Triton injection no other cell organelles were altered. This could indicate differences in membrane sensitivity, which have been reported elsewhere (Wcissman et nl., 1969), or rather be the result of the higher detergent concentration inside lysosomes, where it accummulates (Wattiaux et al., 1963). Production of severe mitochondrial damage when Triton WR-1339 was added to the incubation medium of animal cell cultures was recently reported (Zimmerman et al., 1969). In our completely different experiment,al conditions this finding was not confirmed. Determination of mitochondrial enzymes did not show evidences of mitochondrial membrane damage, except for a slight increase in soluble glutamic dehydrogenase at 24 hours. High values of soluble succinic dehydrogenase in all groups were probably due to the technic employed, which is not completely specific for this enzyme and measures the presence of other oxidizing systems (Green et al., 1965). The variations in the total activity of acid phosphatase and glutamic dehydrogenase observed in some periods might be due to a direct effect of Triton. These effects have been described for other enzymatic systems in vitro using Triton X-100 (Gutman, 1970), but they would not influence enzyme distribution. Mitochondrial integrity was also supported by ultrastructural studies. From 12 hours after Triton administration a significant rise in serum sorbitol dehydrogenase was observed, probably reflecting a cell membrane damage. Increased permeability of liver cell membrane, with elevation of intracellular enzymes in serum, in t,he absence of necrosis, is a well known fact (Henley, 1959). In our experiments it occurred without signs of ultrastructural changes and progressed up to a level which is below those exhibited by cases with overt necrosis, like CC14 or thioacetamide intoxication (unpublished observations). Sinusoidal pole alterations have been emphasized by some authors as an important event in some cases of necrosis (Schaffner, 1966). It might be suggested, therefore, that they represent a contributory factor in the Triton-induced aggravation of liver cell necrosis. That liver cell membrane injury is a direct effect of Triton cannot be estab-

360

HER?;JN

ITURRIAGA

ET

AL.

Iished, since it appeared clnwnologically after the lysoromal damage was produced. It has been rrportetl t,h:lt, 1ysosom;tl injury may induce cc>11nltcrations (,4llison, 1966) and in sonw casev may crew lcnd t#o cell deat)h (Weissmann, 1967). It could be 1~ostulatecl then that tlw ~11 membrane damage produced 1)~ Triton is secondary to the Iysosomal injury. Furt,her studies are, however, necessary to elucidate this problem. ACKNOWLEDGMEIVIY The authors are gratc>ful to Dr. Guillermo Ugartt: for his helpful Dnza and Mr. Jorge Cnsano\-a for their technical help.

adviw.

and

Miss

Margarita

REFEREYCES C. (1966). The possible role of lysosomes in carcinogen&s. hoc. Roll. &x. died. 59: 56&871. ASADA. M., and GALAMBOB, J. T. (1963). Sorbitol dehydrogennse and hepatocellular injury: an experimental and clinical study. Grrsfroenterology 44: 57%58i. FARBER, E., SHULL, Ii. H., VILLA-TREVINO. S.. TIom.~~~~. B., and THon1.m. M. (1964). Biochemical pathology of acute hepatic adenosine-triphosphate deficiency. A$rrrfure Lo?zrlon 203, 34-40. FISKE, C. H., and SUBBAROW, Y. (1925). Calorimetric determination of phosphorus. J. Biol. Chem 66,375-400. FHIEDWAN, M.. and BYERS, S. 0. (1957). Mcchnnism underlying Il~l,rr~holestcrol~~min induced by Triton WR-1339. Amer. J. Physiol. 190, 43s-445. GARNER, R. C., and MCLEAN, A. E. M. (1969). Incrrased susceptibilityto carbon tetrachloride poisoning in the rat after pretreatmrnt with oral phcnobarbitonn. Biochem. Phrtrmacol. 18, ALLISON,

645-650. D. E., MII. S.. and KOH~X~T. I. Succinic deh>.drogc~nnw. GUTMAX, M. (1970). Effects of Triton Chem. Phys. 2, O-14. HENLEY, K. S. (1959). The transnminaw GREEN,

system.

P. M. (1965). Studies 1. Biol. (‘hem. 217, X-100 on mitochondrinl content

on the

trrminal

electron

transport

551-567.

of parcnch>-matolls

N.kDH liver

dchydrogennse cells.

Physiof.

Gnstroetzferology

36,1-6. H.. POSALAI(I, I., and RI-HIP;, E. (1969). dggmvation of hr~llntic nerrosis by lysosomnl injury. Exp. Mol. Pnthol. 10, 231-239. Jun.4H. J. D. (1969). Biochcmicnl disturh:mc*cs in liver injury. Uril. llletl. Bull. 25, 274-377. LUFT. J. H. L. (1961). Improucments in epoxy resin embedding methods. J. Biophus. Biochem. Cyfol. 9,409-414. MASNOW. A. T.. and J,II,LIG. R. D. (1954). “Histoputhologic Technic and Prwiiwl Histochemistry.” pp. 117-118. Blnkiston. New To&. MCLEAN. A. E. M., M~Jx~N, R.. and JUDAH. J. D. (1965). Cellular necrok in (hrx li\-er inducrd and modified hy drugs and other agents. Itit. IZev. F:xp. Prtlhol. 4, 127-157. RXYXOI,I~S. R. S. (1963). The 11s;~ of lwd ci trnte at. high pH as an cl~c~tron-olr:lcl~~c *t:Gn in rlwt,ron microscopy. J. Cell Biol. 17, 205-212. R,oe. H. J.. EPRTEIX. J. H.. and GOLUSTEIN. N. P. (1949). ;I p~otomeiric* mr~thod for t11~ delcrminntion of inulin in plnsma and urine. J. Bid. Chern. 178, 539-845. SCASU. A.. ORIRXTE. I’.. RZ.i,JEWSKI, J. 31.. McCo~u~~.~cs, I,. J., and P;~c;I.:. I. H. (1961). Triton hyperlipclmia in dogs. II. ~~thcrosclrrosis. diffusr Iipidosis and dcpl(-tiun of fat stores prodllccd hy prolorrgrd :~tlnlinirtr:ttiotl of t.hcx non ionic, ,~llrf:l~r,-:~l.ti~(~ :Igcn( /. 1$x/). .~fprl. 114, 279-294. SCHAFFSER. 17. (1966). Intralobul:u cnhangcs in 1lrpatocyte.G and ihc electron microscopic mescnchymal response in ncutc, viral hrpatitis. Nedici,ie 45, 547-552. PLATER. 33. C.. and BOKXKR. 147. D. (1X22). TIx rffpct of flllorirle on tllp succinic oxidase system, Biochena. J. 52, 185-196. ITURHIAGA.

LYSOSOMAL

INJURY

AND HEPATIC

NECROSIS

361

R., WIBO, M., and BAUDHUIN, P. (1963). Influence of the injection of Triton WR1339 on the properties of rat liver lysosomes. In “Lysosomes,” pp. 176196. Ciba Found Symposium. Little, Brown, Boston, WEISSMANN, G. (1967). The role of lysosomes in inflammation and disease. Ann. Rev. Med. 18, 97-112. WEISSMANN, G., HIRSCHHORN, R., and KRAKAUER, K. (1969). Effect of mellitin upon cellular and lysosomai membranes. B&hem. Pharmacol. 18,1771-1775. ZIMMFXMAN, E. M., VAITUZIS, Z., and HETRICK, F. M. (1969). Mitochondrial damage and inhibition of respiration in animal ceil cultures treated with Triton WR-1339. J. Cell Physiol. 74,67-76. WATTIAIJX,