Effect of Two Gold Compounds on Lysosomes

Ann Rheum Dis: first published as 10.1136/ard.47.6.509 on 1 June 1988. Downloaded from

Annals of the Rheumatic Diseases, 1988; 47, 509-514

Effect of two gold compounds on lysosomes

PERNILLE M0LLER GRAABIEK AND SUSANNE M0LLER PEDERSEN From the Institute of Cell Biology, Department of Anatomy, University of Aarhus, Aarhus; and the Department of Clinical Chemistry, Svendborg Hospital, Svendborg, Denmark

SUMMARY The effect of two gold(I) compounds on stability of lysosomes in vitro was studied. Lysosomes from homogenates of rat kidney cortex were isolated by differential centrifugation. These lysosomes were incubated at 37°C with widely varied concentrations of sodium aurothiomalate and sodium aurothiosulphate for 5, 35, and 65 minutes. Acid phosphatase activities were measured and used as an indication of lysosomal membrane stability in the presence and absence of drugs. The enhanced release of acid phosphatase from lysosomes by aurothiomalate and aurothiosulphate was related to dose, but the drugs differed substantially in their potencies. The disruptive effect on lysosomes was more marked for aurothiosulphate than for aurothiomalate. In addition, both drugs inhibited acid phosphatase activities at relatively high gold concentrations. Aurothiomalate had a moderate and aurothiosulphate a weaker inhibitory effect on the enzyme. Our results indicate that aurothiomalate and aurothiosulphate exert their beneficial effect in the treatment of rheumatoid arthritis through mechanism(s) other than lysosomal membrane stabilisation. copyright. Key words: gold thiomalate, gold thiosulphate, acid phosphatase.

The effectiveness of gold compounds in the treat- chrysotherapy, which makes it possible that gold ment of rheumatoid arthritis has stimulated interest compounds exert their therapeutic or toxic effects, http://ard.bmj.com/ in the mechanism by which these drugs exert their in some way, on the lysosomes. therapeutic effect. The aim of the present study, therefore, was to A function which has been attributed to other investigate the effect of various concentrations of anti-inflammatory drugs, steroidal and non-steroidal, gold on a well characterised lysosomal fraction and is that of lysosomal membrane stabilisation." This to compare the effect of two different gold com- concept implies that these drugs prevent the labilisa- pounds in order to elucidate the role of the ligand. tion of lysosomes and thus inhibit the liberation of deleterious lysosomal enzymes, which are capable of Materials and methods on September 24, 2021 by guest. Protected degrading the various components of connective tissue. It has often been suggested that gold com- DRUGS pounds may also act by stabilising the lysosomal Sodium aurothiomalate was purchased from Rhone- membrane. The few studies taking this approach are Poulenc Pharma Norden, Birker0d, Denmark and conflicting, however. No stabilising effect of gold sodium aurothiosulphate was obtained from Ferro- thiomalate and gold thioglucose on rabbit liver san, S0borg, Denmark. Gold stock standards of lysosomes5 and of gold thiomalate on rat liver aurothiomalate and aurothiosulphate were prepared lysosomes2 in vitro was found, whereas a stabilising in 0-25 M sucrose, pH 7-2, in the concentration effect of gold thiopropanol sulphonate on rat kidney range 1-25-125 ,ug gold/ml and 1-25-250 ,ug gold/ml lysosomes was observed.6 respectively. The concentrations of aurothiomalate It has been found that gold accumulates in the and aurothiosulphate are expressed as elementary lysosomes of synovial cells7-9 and renal proximal gold throughout. cellsl0 in patients with rheumatoid arthritis during PREPARATION OF LYSOSOMAL SUSPENSION Accepted for publication 26 November 1987. Four non-starved male Wistar rats, weighing Correspondence to Dr Pernille M0ller Graabek, Anatomisk 250-300 g, were used in each experiment. The Institut A, Aarhus Universitet, DK-8000 Aarhus C, Denmark. animals were anaesthetised by intraperitoneal 509 Ann Rheum Dis: first published as 10.1136/ard.47.6.509 on 1 June 1988. Downloaded from

510 Graabxk, Pedersen injection of sodium pentobarbital (50 mg/kg body propyethylene test tubes containing 700 itl of 0-25 M weight) and the kidneys perfused in situ with ice sucrose, pH 7-2, at 0-40C, without (controls) or with cold 0-3 M sucrose containing 1 mM edetic acid, pH gold added. The final concentrations of gold in the 7-2, for three minutes. The kidneys were removed, lysosome-aurothiomalate suspensions were 0-2, 04, the renal cortexes rapidly excised, weighed, minced, 1, 2, 4, 10, 20 sg/ml and in the lysosome-aurothio- and diluted 1:10 (w/v) with the ice cold solution sulphate suspensions 0-2. 0-4, 1, 2, 4, 10, 20, 40 used for perfusion. The tissue suspension was then tg/ml. Two samples (0.5 ml) of each mixture were homogenised in a Potter-Elvehjelm glass homogen- preincubated at 37°C for exactly 5, 35, and 65 iser by 10 complete strokes with a Teflon pestle minutes, after which enzyme substrate was added rotating at about 200 rpm. Care was taken to follow for measurement of the free acid phosphatase the same homogenisation procedure and to use the activity. same Teflon pestle and glass homogeniser for all Samples (0-5 ml) of controls and lysosomal experiments in order to reduce variations in the suspensions containing gold were frozen (in liquid degree of lysosome disruption. C02) and thawed three times for complete disrup- The homogenate was fractionated by the method tion of the lysosomes. The samples were then of Maunsbach." The light fraction and the large preincubated for 35 minutes at 37°C before enzyme mitochondrial fraction were carefully removed from substrate was added for determination of the total the materials sedimenting at 9000 g and the remain- lysosomal acid phosphatase activity. ing pellet enriched with lysosomes (primary granule The samples were not centrifuged before assay of fraction) was gently rinsed and resuspended in 30 ml the released acid phosphatase in order to eliminate ice cold 0-25 M sucrose containing I mM edetic acid, the errors due to differences in the sedimentation of pH 7-2. This lysosomal suspension was used in the free lysosomal enzymes or gold compounds, or following experiments. Further purification of the both, with the lysosomal membranes. lysosomes as described by Maunsbach" (e.g., re- moving the last relatively small amount of mitochon- ASSAY OF ACID PHOSPHATASE copyright. dria) was found to be unnecessary in this study, Acid phosphatase was assayed at pH 5-0 with whereas the time saved by stopping the isolation at 13-glycerophosphate as substrate. Liberated phos- the stage of primary granule fraction was considered phate was determined by the method of Lowry and of importance. Lopez14 using amidol as reducing agent. The specific acid phosphatase activity was expressed as tmol of CONTROL OF THE LYSOSOMAL FRACTION phosphate liberated/20 min/mg protein. Protein was For electron microscopic analysis of the composition determined by the method of Lowry et al'9 with http://ard.bmj.com/ of the lysosomal fractions pellets enriched with bovine serum albumin as a standard. For reasons of lysosomes were fixed in situ in the centrifuge tube or comparisons the activity of acid phosphatase is fixed in suspensions and further prepared for expressed as percentage of control total enzyme electron microscopy as described by Maunsbach" activity. and Sjostrand and Borgstrom.'2 Ultrathin sections, EFFECT OF GOLD ON ACID PHOSPHATASE perpendicular to the surface of the pellets and from ACTIVITY different parts of them. were cut through their entire The effect of gold on the total acid phosphatase on September 24, 2021 by guest. Protected depth. The sections were stained with uranyl acetate activity of a disrupted lysosomal suspension was and lead citrate and examined in a JEM-IOOB studied. Aliquots (0-4 ml) of a lysosomal suspension electron microscope. were transferred to test tubes and 100 ,ul 0-25 M Biochemical determination of total acid phospha- sucrose, pH 7-2, without (controls) or with gold was tase activity and cytochrome oxidase (mitochondrial added at different steps in the procedure: (a) before marker enzyme) activity in the lysosomal suspension the lysosomal suspensions were frozen and thawed, was compared with that of the homogenate. Cyto- (b) after freezing and thawing, and (c) after incuba- chrome oxidase activity was measured by the tion for acid phosphatase before determining the method of Wharton and Tzagoloff.'3 liberated phosphate. The final gold concentrations for the lysosomal suspensions incubated with ASSAYS OF LYSOSOMAL MEMBRANE aurothiomalate were 0-2 and 20 ig/ml and for STA B I LITY aurothiosulphate 0-2, 20, and 40 [tg/ml. Labilisation or stabilisation of lysosomal membranes *in the presence of gold was ascertained by comparing STATISTICAL ANALYSIS the release of the lysosomal marker enzyme, acid Statistical analysis was by Student's t test with phosphatase, with that in controls. Aliquots (3-5 ml) significance level at p

Effcto[t/o gold cOmpOlunlsloSoi lsVosomIUS 11

Results Oving to the direct inhibitorx effect of -old on lxsosomal acid phosphatase it xas found necessarx PROPERTIES OF 1 HE l Y SOSOI AI ERR ATION to determine the total acid phosphatase actixitv in When analysed bx electron microscopv the lvso- all lsosomal1 suspensions. Fig. 1 shoxxs the effect somal fraction contained predominantlx membrane of various concentrationis of aurothiomalate anid limited bodies (0(5-1 5 Ltm in diameter) with exen aurothiosulphate on lysosorrmal acid phosphatase. densely stained contents. Mitochondria and some Acid phosphatase actix itv xvas reduced bv 8"O. 30%. unidentified membranous structures xvere calso pre- and 66"0) when preincubated with aurothiomallate at sent but in considerablv less numbers than the 4(0. 10. and 20 Um/ml respectixely. A much wxeaker lvsosomes. Biochemicallv. the lvsosomal fraction contained about 7 50O of the acid phosphatase actixitx of the A UROTHIOMALAT E 5 MIN homogenate and the specific actixitx of this enzxme 100 O ENZYME RELEASE (ACTUAL) \was eight to nine times greater than in the homotTen- U ENZYME RELEASE (CORRECTED) ate. In contrast. only about 0(2"/ of the cxtochrome oxidase activitv of the homooenate w,as present in this fraction. and it had a low specific actixity. The ultrastructural and biochemical data thus demon- 50 ~ strate a lysosomal fraction quite rich in lysosomes. though still contaminated bx other cell components. 11111111~~~~~~~~~~~~

EFFECT OF GO D ON ACTIVIT\ OF ACID PHOSPH A TASE FROM R AT KIi)N EY -lm 3 MINlm L S O S OM1 S 35 MIN The effect of oold on the total cacid phosphatase U- 100 - copyright. Llu activitv of a disrupted lysosomal suspensioil xhen Ll gold w as added at different steps in the procedure as described under 'Materials and methods xwas a- studied. The results showed that aurothiomralate had 0 z a moderate and aurothiosulphate a xweaker in- cU 50-* hibitorv effect on the lvsosomal enzxme. Gold did 0O 50 http://ard.bmj.com/ not affect disruption of the lvsosomes or determin- C-) 0 ation of liberated phosphate. 4 - LL 0 ° 0O _

Ur) U

o-L<0LL a_ Z 0 50

0 0.2 04 1 2 4 10 20 0 0.2 0.4 1 2 4 10 20 40 ,ug Au/ml ,ug Au /ml Fig2. 2 EtffCct ot aurothionila/te at the rclct/ea afacid Fit. I Direct ftf''cctotaii(rotlioia/icltitt, tandtl ph)o/sp/hatase frowit(it kidtnecy I/vsosoatnw. Lv.mso%lnes i etc aurot/iosuilphate oan aicid phosphatase tr0-on disrupted preincahated ait 37 (C itllt/i (iartllinalitltt ti tlic 1/v.soso;na/l. tsapenh.oti.%. Eaichi a/aci represents t/it 11101/ii concciitratioan and(i far the titlne ptriods idicated. Each (SD) aft ti/ t alit (ictiic ii at acidIphosaphatasc iii tllc i'a/laic rtiptnt% thef mean(.SLe ) ilothat cscparatc lxS'C)5'(oai1a1/ ~asl(c1itanliscd inI i/ic cxperillitelti s%/IC1oi' inl cxpe)rinctnits. Significanit at p

512 Gi,aabVek Pedersen inhibitory effect of aurothiosulphate on acid phos- A lrothliotnalakte phatase activity was observed namel, 10°/ at 20 When intact lysosomres were preincubated at 37 C sg/ml and 19% at 40 Ltg/ml. for 5. 35. and 65 min with aurothiomalate in concentrations ranging from 0-2 to 2()0g/ml the EFFECT OF GOLD( I) COMPO UN DS ON lIsosomal enzvme actiVitv was found to decrease STABII1 IT' OLF I ASOSOML S ROM RiNT gradually with concentrations >10 Lg/ml (Fig. 2). K I D N E Y This may be thought to indicate a stabilising effect of Figs 2 and 3 illustrate the effect of aurothiomalate aurothiomalate on the Ixsosomes at high gold and aurothiosulphate on the release of acid phos- concentrations. This apparent stabilisation effect ot phatase from rat kidney lvsosomes. It was found aurothiomalate on the lysosomal membrane. that gold had a dose dependent labilisation effect on however. could be accounted for entirelv bv a direct the lysosomal membrane and this effect was more inhibitory effect upon the enzvme activitv. Thus marked for aurothiosulphate thani for aurothiomaflate. corrected for the observed inhibitorv effect upon the acid phosphatase activity the results for five minutes of preincubation showed a gradual labilisation AUROTHIOSULPHATE 5 MIN effect of aurothiomalate at concentrations >1 )0 100 0 ENZYME RELEASE (ACTUAL) * ENZYME RELEASE (CORRECTED) ltg/ml. It appeared. however, that the enzyme was slightlyx activated at small concentrations of aurothiomalate (Fig. 1). When corrected for this effect the release of acid phosphatase fell below the 50- control value, indicating a wevak lvsosomal membrane stabilising effect (Fig. 2). No statisticallx significf1ant effect of aurothiomalate on the lysosomal membrane could be shown at the 3-5 and 65 minute copyright. >- 0 time intervals. > ~~~~~~~~~~~~~35MIN bbb b b bb b b LJL) 100 b Aurothi)osuilpliate Cf)< When lysosomes were preincubated with auro- 4~-0J thiosulphate at 37°C for five minutes the release of acid phosphatase was increased in the concentration range tested, reaching a maximum at 10 stg/ml (Fig. 0 5 3). When corrected for the observed direct inhibi-http://ard.bmj.com/ 4a- torv effect of aurothiosulphate on acid phosphatase a I-L activitx gradual labilisation effect of the drug over 0 the entire concentration range (0-'-40 .tg/ml) was -. 0 observed. A dose related effect could also be shown after the 35 minute period, whereas no effect of the drug could be demonstrated after 65 minutes

(Fig. 3). on September 24, 2021 by guest. Protected Discussion A well defined lysosomal fraction from rat kidnev cortex was incubated at 37°C in the presence of widely varied concentrations of aurothiomalate and aurothiosulphate. The chosen concentration range

0 0.2 0.4 1 2 4 10 20 40 of gold included the therapeutic levels (0.3-5 ig/ml) ,ug Au/ml achieved in clinical use of these compounds. 6 17 The present study showed that the two gold com- Fig. 3 Effect ofaurotliosuliphate on the release of acid pounds enhanced the release of lysosomal marker phosphatase from rat kidnesv lsosomes. LYsosomes were enzyme in a dose related fashion, and that they preincubated at 37 C with aurothiosulphate at the differ in their potencies. At a concentration of 20 Mtg and the time indicated. Eaclh concentrations for periods gold/ml aurothiomalate and aurothiosulphate en- value represents the mean (SD) offour separate hanced the release of acid phosphatase by 10% and experiments. "Significant at p<005 v control: "significatit at that p

Effect of two gold compounds on lysosomes 513 membranes than aurothiomalate. In addition, the gold compounds may inhibit lysosomal phospholi- two drugs had a direct inhibitory effect on acid pases and thereby decrease the transport of amino phosphatase, with aurothiomalate being the most acid hydrolysis products out of the lysosomes.23 potent inhibitor. Aurothiomalate showed 55% and Accumulation of hydrolysis products could lead to aurothiosulphate 10% inhibition of enzyme activi- osmotic swelling and eventually disruption of the ties at 20 ,ig gold/ml. The present data demonstrate lysosomes, resulting in enhanced release of acid that it is important to correct for the interference of phosphatase. This mechanism of phospholipase gold with enzyme activities to obtain a valid inhibition has been described for various cationic interpretation of the experimental results. drugs and for other metal ions.24 The present labilising effect of high concentra- A possible mechanism of action in vivo consistent tions of gold on isolated lysosomes has never been with the present findings is that aurothiomalate and described before. In the few previous studies on the aurothiosulphate, which are anionic compounds, effect of gold on the stability of lysosomal mem- bind protons in the acidic interior of lysosomes. This branes in vitro no effect of aurothiomalate was would raise the intralysosomal pH and thereby found.2 5 Our findings are in general agreement neutralise the lysosomal enzymes, which if released with the observations that aurothiomalate'8 and could perpetuate the inflammatory process. In aurothiosulphate'9 at low concentrations do not addition, it is probable that the gold compounds affect the release of lysosomal enzymes from affect lysosomal function or intracellular processes, polymorphonuclear leucocytes, whereas auro- or both, by a direct inhibitory effect on lysosomal thiosulphate'9 at higher concentrations shows a enzymes as suggested by Persellin and Ziff,25 Ennis slight inhibitory effect. As the authors'9 did not et al,5 and Ghadially et al.7 This hypothesis is in investigate the direct effect of gold on the activities accordance with our findings and the findings of of lysosomal marker enzymes it is impossible to others5 21 25 that gold at high concentrations has a decide whether the inhibitory effect observed is due direct inhibitory effect on lysosomal enzymes. to a stabilising effect of gold on the lysosomal Owing to the high concentrations of gold required to copyright. membrane or simply due to an inhibitor effect on produce significant inhibition of lysosomal enzymes the activities of the lysosomal enzymes as happened it is unlikely that gold exerts an extracellular effect to be the case in our study. on lysosomal enzymes in vivo. High gold concentra- The different ability of the two gold compounds to tions within lysosomes could, however, readily be labilise the lysosomal membranes and to inhibit the attained. It has been found that synovial cells after activity of acid phosphatase strongly suggests that chrysotherapy show large lysosomal bodies (auro- the interaction of gold with lysosomes is affected by somes) containing numerous lipid droplets,7 myelin http://ard.bmj.com/ the ligand. This idea is supported by the findings structures,26 and gold deposits, which provides that auranofin shows a potent inhibitory effect,'9 further supportive evidence for the intralysosomal whereas aurothioglucose even at high concentra- action of gold compounds on catabolic enzymes. tions shows no inhibitory effect on the release of In summary, aurothiomalate and aurothiosulphate lysosomal enzymes from polymorphonuclear have been shown to labilise lysosomal membranes at 5 20) and by the findings that different relatively high gold concentrations, and, in addition,

antiarthritic gold preparations differ markedly in it was found that the drugs exert a direct inhibitory on September 24, 2021 by guest. Protected their ability to inhibit the activity of lysosomal effect on acid phosphatase-the lysosomal marker enzymes. enzyme. Although it is not possible to determine Although the present study cannot explain how directly from these in vitro experiments what the in the two gold compounds exert their labilising effect vivo effects of gold compounds may be, our results on in vitro lysosomes, one or more of the following do not support the idea that gold compounds exert possibilities shoulti be considered: (a) the gold their beneficial effects by stabilising the lysosomal compounds may interact with the outer or inner membranes. It appears more likely that these drugs surface of the lipoprotein-membrane structure and act by inactivation of lysosomal enzymes by inducing thereby change the integrity of the lysosomal a raised intralysosomal pH or by a direct inhibitory membrane, resulting in an enhanced release of acid effect on lysosomal enzymes, or both. The differences phosphatase; (b) the gold compounds may accumu- observed in the effects of the two gold compounds late within lysosomes by a concentration dependent on lysosomal membranes and lysosomal acid phos- mechanism or a proton pump dependent mechan- phatase suggest that their individual ability to ism, or a combination of both, as described for weak interact with the lysosomal system is affected by the base amines.22 The accumulation of drugs could ligand. lead to osmotic swelling and even disruption of lyso- The authors are indebted to Mrs Hanne Weiling for excellent somes with release of acid phosphatase; and (c) the technical assistance. Ann Rheum Dis: first published as 10.1136/ard.47.6.509 on 1 June 1988. Downloaded from

514 Graabxk, Pedersen

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cells of the rat intestinal epithelium during fat absorption. J cytochemistry of rheumatoid synovial membrane with special http://ard.bmj.com/ Ultrastruct Res 1967; 20: 140-60. reference to lysosomes. Ain J Pathol 1964; 44: 853-66. on September 24, 2021 by guest. Protected