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Lysosomal stabilizing effect of chlorphentermine in the rabbit alveolar macrophage
Got Ph.rmm 'Jo[ 10. pp ~ to 8~ P e r g a m o n Press l i d 1'~79 Printed in Great Britain
0306-3623 79 (13111-0083Stl2(~)0
L Y S O S O M A L STABILIZING E F F E C T OF C H L O R P H E N T E R M I N E IN THE RABBIT ALVEOLAR M A C R O P H A G E MARK J. REASOR and ELIZABETH R. WALKER Department of Pharmacology and Department of Anatomy, West Virginia University Medical Center, Morgantown, WV 26506, U.S.A. (Receit'ed 13 July 1978)
Abstract--l. Rabbits were injected (i.p.I daily with chlorphentermine 130mg;'kg), 5 days a week for 7 weeks. 2. Alveolar macrophages were collected from the lungs of control and treated rabbits by pulmonary lavage. 3. When compared to controls, the latencies of the lysosomal acid hydrolases, acid phosphatase and fl-N-acetyl glucosaminidasc were increased in lysosome-containing fractions from alveolar macrophages of treated rabbits. The same results were obtained when chlorphentermine was incubated in vitro with a lysosomal fraction from macrophages of normal rabbits. 4. The increased latency of lysosomal enzymes following chlorphentermine treatment can be explained by the drug's ability to stabilize lysosomal membranes.
in 0.154 M NaCI 5 days a week for 7 weeks. Control rabbits received an equal volume of vehicle. Because chlorphentermine is an anorectic drug, treated rabbits initially lost weight, recovered and proceeded to gain weight although at a rate less than non-injected controls. Therefore in this study, control rabbits were pair-fed to ensure weight gains comparable to the treated animals. One control and one treated animal were sacrificed on the same day, macrophages collected and the experiments performed.
INTRODI.'CT1ON The administration of a large number of amphiphilic drugs leads to a drug-induced phospholipidosis in many organs of experimental animals (LiJllmann et al., 1975). The disorder manifests itself within the lysosome of the cell resulting in the formation of lamellated lipid inclusions which have been termed "myeIoid bodies" (Abraham et al., 1968: Hruban et al., 1972; LiJllmann et al., 1975; Drenckhahn et al., 1976: St~iubli et al., 1978). Although these ultrastructural alterations have been described extensively, very little information exists as to specific effects on lysosomes which might lead to functional alterations in this organelle. St~iubli et al. (1978), have reported that treatment of rats with the amphiphilic drug maprotiline results in a labilization of hepatic lysosomes. We have administered chlorphentermine, another amphiphilic drug used commonly to induce this disorder. to rats, and observed changes in lysosomal properties of alveolar macrophages (Reasor et al., 1978a). In addition to resulting in an increase in activity of lysosomal acid hydrolases, treatment with chlorphentermine resulted in alterations in the integrity of the lysosomes as measured by the latencies of two acid hydrolases, acid phosphatase (AP) and fl-N-acetyl glucosaminidase (flNAG). The latency of AP increased while that of flNAG decreased. In an effort to extend our studies to other species, we examined the effects of chlorphentermine treatment on lysosomal properties of the alveolar macrophage of the rabbit, an animal in which drug-induced phospholipidosis has not been well documented. This report presents the results of this investigation. MA'I'ERIAI^S A N D
Collection of alveolar macrophages
Some time after the last drug administration (24 hr). the rabbits were deeply anesthetized with pentobarbital (50 mg/kg). The abdomen was opened by a midline incision and the animals exsanguinated by severing the abdominal aorta. The thorax was opened along the sternum and the lungs and trachea were removed intact. They were rinsed liberally with cold 0.154M NaCI and trimmed of extraneous tissue. The excised lungs were lavaged via the trachea with cold calcium and magnesium-free Hank's Balanced Salt Solution (HBSS), pH 7.4. Approximately 40ml were needed to fill the lungs, and the fluid was allowed to drain out by inverting the lungs. Approximately 250ml of fluid was collected, beyond which virtually no additional cells were recovered. Macrophages were recovered from the lavage fluid by sedimentation at 5000 for 10rain, washed twice and resuspended in complete H BSS.
METHODS
Animals and dru(.l administration
Chlorphentermine hydrochloride (Warner Chilcott, Morris Plains. N J) was administered to adult, male New Zealand white rabbits (2-3 kg) at a dose of 30mg/kg, i.p.. 83
Assays Protein content of the cells was measured according to the procedure described by Hartree (1972) using bovine serum albumin as standard. The following enzyme assays were performed on macrophages in the presence of 0.10,. Triton X-100:acid phosphatase -with p-nitrophenyl phosphate as substrate (Turnbull & Nell, 1969); /~-glucuronidase- with p-nitrophenyl-fl-o-glucuronide as substrate (Lockard & Kennedy. 1976): aryl sulfatase- with nitrocatechol sulfate as substrate (Dodgson et al.. 1955); flgalactosidase--with p-nitrophenyl-fl-D-galactoside as substrate (Levy & McAIlan, 1963), fl-N-acetyl glucosaminidase --with p-nitrophenyl-N-acetyl-fl-D-glucosaminide as substrate (Sellinger et al.. 1960): and a-mannosidase--with
84
M-XRK .I. RI',SOR and EII:Z.~BITII R. W.'~IK|R
p - n l t r o p h e n } ]--/-i)-nlanllosld¢ a~, '-;ubMrale I( "Ollchl¢ & H a } . 1959)
.'~lcasurement o! httencv As a measure of I.,,sosomal menlbrane stability, the latenties of .~,1~ and /~NA(-I ,acre determined. A bsosome-conraining fraction llS.0(~)g pellet) was prepared from homogeni/ed cells IHurst cta/.. 19701 and suspended in 11.25 M sucrose-20 m M Tris-acetate. ptl 7.4. All aliquot of this susp e n s , m 10.9 nail was added to I.I ml of complete HBSS at 37 (" and an aliquot removed immediately to determine baseline hydrolase release. The percent of enzyme activity released at this "'zero time" was measured by centrifuging a portion of tile aliquot sit 15.0(~) g h~r 20 mill and comparing tile non-sedimcntable activity with the total enzyme activity in the aliquot. The remainder of the sample was incubated [or 15ram at 37 (" and the percent of the enz3me acllviL', released into the non-sedimentabie form during this lime was determined as described. The latenc.~ of the enzyme activity was determined b', comparing the percent of the total ac~vit.~ released in 15rain with the baseline release. Macrophages were processed for electron microscopy as described b~ Pine & Lov, [1975). Red hh,od cell (RBC) lysts Blood was collected from tile central ear artery of rabbits b~ drainage through a 20 gauge needle into a heparinized tube. RBCs ~ere sedimented at 1000g for IOmin. washed twice and resuspended ill a medium containing 5 m M KCI. 5 m M glucose. 5raM III=.PES buffer, and 0.154M NaCI. pH 7.4. An equal vohmle of this medium containing chlorphcntcrmme Idouble the assay concentration) and xar.',ing concentrations of NaCI was added to the RBC suspension to gi~e the desired drug concentration and osrnolarit',. Incubation was carried out for 15min at 3"7,(" An aliquot `aas then remo,,ed and the RB('s sedimented at 10Ollq for 1 0 r a m The supernatant phase ',',,is rem
W h e n c o m p a r e d to controls, a slight but nonsignificant increase was o b s e r v e d in the n u m b e r of m a c r o -
Table I. Effect of chlorphentermine adminislration on the recovery of alveolar macrophages ['rolT1 rabbit lung,, b', pulmonary la~age
Macrophages
("ells kg b.`a. I× 10 I
mkz Protein Ill- Ceils
('ontrol
2.67 4- 11.61"
1.80 ± (I. It)+
Treated
3.61 ± 0.52
2.(12 + 024
Rabbits i.p.I for 7 * Mean .t- Mean
were treated ~,ith chlorphcntermine 130nlg kg weeks. 5 da~s per week. 4: S.t-.M. [N - 71. Jr S.E.M. IN = 4i.
p h a g e s recovered from the l u n g s of treated rabbits (Table 1). T h e p r o t e i n c o n t e n t per 10" cells was not significantly different either. Electron m i c r o g r a p h s ~not s h o w n ) o f alveolar m a c r o p h a g e s from the d r u g - t r e a t e d a n i m a l s failed to show the characteristic accumulation of lysosomallyderived lamellated inclusions w h i c h are a s s o c i a t e d with c h l o r p h e n t e r m i n e a d m i n i s t r a t i o n in o t h e r species ( L i J l l m a n n - R a u c h et al.. 1972: L i i l l m a n n - R a u c h & Rei[, 19741. T h e similarity of t h e u l t r a s t r u c t u r e of the m a c r o p h a g e s from the c o n t r o l a n d c h l o r p h e n t e r m i n e treated r a b b i t s indicated that s e v e n weeks of d r u g t r e a t m e n t h a d failed to i n d u c e p h o s p h o l i p i d o s i s as .judged by a m o r p h o l o g i c criterion. Since t h e l y s o s o m e is n o r m a l l y t h e first o r g a n e l l e visibly affected in this d i s o r d e r ( L i i l l m a n n et al., 19751 it was of interest to d e t e r m i n e w h e t h e r functional c h a n g e s h a d o c c u r r e d within t h e l y s o s o m e w i t h o u t the d e v e l o p m e n t o f t h e lamellated inclusions. T h e activities of six l y s o s o m a l acid h y d r o l a s e s were m e a s u r e d a n d f o u n d not to differ in the two g r o u p s w h e n e x p r e s s e d as a f u n c t i o n o f cell n u m b e r or cell p r o t e i n i T a b l e 2). L y s o s o m a l m e m b r a n e stability is a n i m p o r t a n t d e t e r m i n a n t of l y s o s o m a l f u n c t i o n w i t h i n t h e celL As a m e a s u r e of m e m b r a n e stability, t h e latencies of two l y s o s o m a l acid h y d r o l a s e s , A P a n d /~NAG were e x a m i n e d in alveolar m a c r o p h a g e s following chlorp h e n t c r m i n e t r e a t m e n t (Table 3). F o l l o w i n g 15 m i n of i n c u b a t i o n t h e r e was a 117", increase in t h e release of A P from a l y s o s o m e - c o n t a i n i n g fraction p r e p a r e d from the c o n t r o l m a c r o p h a g e s . In c o n t r a s t , t h e r e was only a 70",, increase in A P release from t h e prepa r a t i o n from cells of treated rabbits. T h i s c o m p a r i s o n indicated that t h e r e was a significant i n h i b i t i o n [P < 0.05) in the release o f A P (increased latency1 in s a m p l e s from the c h l o r p h e n t e r m i n e - l r e a t e d rabbits. A
Table 2. The effect of chlorphentermine administration on lysosomal acid hydrolase activities of rabbit alveolar macrophages
Macrophages
Acid phosphatase
Control Treated
141.0 z 26.5* 159.3 ~_ 34.6
21.9 4- 2.0 24.0 4- 4.3
Control Treated
81.4 ± 18.5 80.6 4- 14.7
12.4 _. 1.7 12.6 _.- 0.9
fl-(ilucuronidase
Aryl sulfatase
/1-Galactosidase
(nmoles.:min 10" cells) 15.5 4- 1.0 58.2 _+ 2.6 18.2 + 2.() 6(I.9 _+ 10.6 {nmoles.'min:mg protein) 8.7 4- 0.6 32.8 _~_ 2.9 9.1 _-2 0.7 29.7 -* 3.4
/'¢-N-Acetylglucosaminidase
:~-Mannosidase
857.8 _+ 82.7 977.6 +_ 141.1
38.4 4T 4.8 37.0 + 8.9
483.6 ± 61.0 481.3 _+ 26.7
21.8 t_ 3.5 17.9 _+ 2.8
Rabbits were treated with chlorphentermine {30mg'kg) for 7 weeks. 5 days per week. * Mean 4- S.E.M. IN = 4~.
85
Chlorphentermine and lysosomes Table 3. The effect of chlorphentermine administration on the latencies of lysosomal acid hydrolases of rabbit al~.eolar macrophages Macrophages
Time of incubation (min)
Acid phosphatase
/3-N-Acetylglucosaminidase
of Total Activity Released) 7.3 + 0.5* 28.4 + 5.7
(".
0 Control 15 0
15.7 + 1.2 45.7 + 5.3 (*117.0 _+ 21.1",,1 (T72.2 _+ 16.0",,) 8.4 _+ 0.6 30.8 _+ 4.2
Treated 15
14.2 _+ 0.9 (]'69.8 +_ 6.5",,)
mine on RBC stability in hypotonic solution was examined. At 10- 3 M, chlorphentermine inhibited the lysis of RBCs as demonstrated by the shift of the lysis curve to the left (Fig. 2). This plasma m e m b r a n e stabilization occurred when RBCs were incubated in media from 174 to 191 m O s M . The dose-response relationship of chlorphentermine and the inhibition of lysis at 191 m O s M is presented in Fig. 3 and demonstrates that under these conditions the drug is an effective inhibitor of RBC lysis, presumably by stabilizing the plasma m e m b r a n e of the cell. Therefore, in two separate systems in vitro, chlorphentermine is capable of stabilizing membranes.
43.7 _+ 3.9 (T45.3 _+ 9.6".)
Rabbits were treated with chlorphentermine 130mg'kg) for 7 weeks, 5 days per week. * Mean + S.E.M. (N = 4).
similar inhibition in the release of flNAG was observed in the same samples from treated animals. Therefore, while not leading to ultrastructural changes within the lysosome, these results indicated that the chronic administration of chlorphentermine had apparently resulted in an increased stabilization of the lysosomal membrane. To examine this further, the direct effect of chlorphentermine on lysosomal enzyme release was investigated (Fig. 1). The incubation of chlorphentermine with a lysosome-containing fraction in vitro led to an inhibition in the release of both A P and flNAG, particularly at the higher drug concentrations. Thus, chlorphentermine has the ability to directly stabilize lysosomal membranes, at least in vitro. To test this hypothesis about m e m b r a n e stabilization using another system, the effect of chlorphenter-
DISCL:SSION One of the most striking changes in rat, mouse, and guinea pig lung tissue following the daily adminstration (for 5 weeks or less) of chlorphentermine is the appearance of an increased number of free cells in the alveoli (Franken et al., 1970; Karabelnik et al., 1974; Liillmann-Rauch & Reil, 1974; Svendsen, 1977: Reasor et al., 1978b). These cells, which appear to be derived from alveolar macrophages, are enlarged and contain lamellated inclusions resulting in their being termed "foam cells" (Karabelnik et al., 1974; Svendsen, 1977). These inclusions have been shown to be lysosomal in origin (LiJllmann-Rauch & Reil, 1974; Liillman et al., 1975) and it is proposed that they arise as a result of the formation of a complex between the parent drug and phospholipids (Liillmann et al., 1975). This interaction prevents the normal lysosomal degradation of these lipids from occurring leading to their accumulation within the cell and the subsequent formation of the lamellated structures. In a study comparing the ultrastructural changes induced by chlorphentermine in several organs and
70 ~ ] 60
~c.d Phosphotose .~- I%- '~cetylqluCOSOm~nlOoSe
50
4O
T.
L.
3O 2O IO
0
10-3
10.4
5xlO"4
5xlO"5
CONC.(M)
Fig. 1. The effect of chlorphentermine on the release of acid hydrolases from lysosome-containing preparations from rabbit alvcolar macrophagcs. A lysosome-containing fraction was prepared from the alveolar macrophages of normal rabbits and the effect of various concentrations of chlorphenterminc on the release of acid phosphatase (clear bars) and fl-N-acetyl glucosaminidase (stippled barsl was determined (see Materials and Methods for detailed procedure). The values represent the percent inhibition of enzyme rclcase (Mean +. SE.M., N = 5) when compared to fractions incubated in the absence of drug. *P < 0.05.
86
MARK J. RLASOR a n d ELIZABI i n R. V~.:AIK I R
I00
80
60
40
20
A
i
~
120
140
160
i
180
"(>'--i- -
0
200
-
~
220
240
260
mOsM
Fig, 2. The effect of chlorphentermine on the lysis of rabbit red blood cells in hypotonic solution. Rabbit red blood cells were incubated for 15 min, 37 (', in media of varying osmolarity and the percent lysis was measured in the absence ( • ) and presence of chlorphentermine II0 ~ M~ t O ). Each point represents the mean of two experiments. See Materials and Methods for media composition and procedure for measurement of lysis, species. Liillmann-Rauch & Reil (1974) reported that after 12- 14 weeks of drug administration to rabbits (at levels comparable to that used in the present study), only a slight increase in the numbers of macrophages were seen in the alveoli. However, foam cells were present at this time. The present study revealed that no increase in cell number, cell size [as measured by protein content, a parameter which correlates with increased cell size in rat alveolar foam cells (Reasor et al.. 1978b and unpublished results)] or cells of a b n o r m a l morphology appeared in the alveoli of rabbits treated daily with chlorphentermine for 7 weeks. The basis for the difference in susceptibility between the rabbit and other species is unknown. One possibility would be differences in the pulmonary uptake and.'or metabolism of chlorphentermine between these species. This notion is speculative at present,
and a more delinitive explanation awaits research in this area. Even though chlorphentermine administration did not result in the characteristic ultrastructural changes in rabbit alveolar macrophages, specific effects on lysosomal properties resulted. A stabilization of the lysosomal m e m b r a n e appeared to occur in these ceils, and evidence was obtained indicating that the drug may exert this effect by interaction with the membrane. Seydel & W a s s e r m a n n (1976) have shown that chlorphentermine binds to phospholipids, thus providing a molecular basis for this hypothesis. The ability to interact with m e m b r a n e s may be a general characteristic of amphiphilic drugs. Chlorpromazine, an amphiphilic drug reported to induce phospholipidosis (Liillmann-Rauch. 1974) binds to membranes (DiFrancesco & Bickel, 1977i with the major
80
T,
U3
I
> J
60
o z
o i-
40
z
2O
10-3
10-4
10-5
CHLORPHENTERMINE
10-6 (M)
Fig. 3. The dose-dependent inhibition of lhe Ivsis of rabbit red blood cells by chlorphentermine. Rabbit red blood cells were incubated for 15min, 37 ('+ in media of 191 mOsM with ",aoing concentrations of chlorphentermine, and the inhibition of lysis was measured and compared to samples incubated in the absence of drug. Values ;ire the Mean _+ S.E.M. with IN) shown at the base of each bar. See Materials and Methods for media composition and procedure fl~r measuremenl oflysis.
Chlorphentermine and lysosomes site being the non-polar moieties of phospholipids. Chloroquine, which also causes phospholipidosis (Abraham et al., 1968; Liillman et al., 1975) has been shown to stabilize lysosomal membranes (Weissmann, 1966) in vivo and in vitro. Rather high levels of chlorphentermine were required to stabilize lysosomes in vitro in the present study, and it can be questioned as to whether such levels would be reached in vivo with this dosing regimen. Since the concentration of drug was not measured in the macrophage, or more specifically in the lysosomal membrane, this information is not available. In fact, to the best of our knowledge, such measurements have never been reported for lipidosisproducing drugs. Liillmann et al. (1975) speculate that since high levels of drugs are usually administered for long periods of times in such studies, concentrations up to 1 0 - 3 M may be reached in certain organs. If the drugs preferentially bind to phospholipids within membranes as shown for chlorpromazine (DiFrancesco & Bickel, 1977), their actual concentrations at these sites may, in fact, be even higher. The functional implications of lysosomal stabilization in the alveolar macrophage must be considered. Corticosteroid administration leads to an increased stability of macropbage lysosomes and consequently to an increased susceptibility to pulmonary aspergillosis in mice (Merkow et al., 1971). Therefore, treatment with other drugs with similar actions may have serious consequences for the host organism if this leads to an impairment in pulmonary defense. The administration of amphiphilic drugs does have physiological significance in vivo as is evidenced by the work presented by Liillman et al. (1975). They found that the formation of paw edema induced by dextran in rats was inhibited by the chronic pretreatment with amphiphilic drugs. Since the lysosomal system participates in this inflammatory disorder, one explanation of the results is that drug administration led to an inhibition of lysosomal enzyme activity. This seems unlikely since no effect was found on the activities of six lysosomal enzymes in the present study. Another, and one consistent with the results presented here. is that the drugs stabilized the lysosomes reducing their involvement in the response. Perhaps the most significant result of this study is that an amphiphilic drug can have effects on iysosomal properties of alveolar macrophages without inducing the formation of lamellated inclusions. Therefore, in addition to ultrastructural changes, additional parameters should be investigated when assessing the effects of amphiphilic drug administration. CONCLUSION The increased latencies of lysosomal enzymes in rabbit alveolar macrophages following chronic treatment with chlorphentermine can be explained by the drug's ability to stabilize lysosomal membranes. SUMMARY
The chronic administration of chlorphentermine to rabbits failed to induce phospholipidosis in alveolar macrophages as judged by the absence of ultrastructural changes within the lysosome of the cell. How~.,.P. 10.'2 a
87
ever, a specific effect on the lysosome was observed following drug-treatment. An increased stabilization of the lysosome resulted as measured by an increase in the latencies of the lysosomal acid hydrolases, acid phosphatase and fl-N-acetyl glucosaminidase. The addition of chlorphentermine to a lysosome-containing fraction led to an increase in the latencies of these two hydrolases, indicating that chlorphentermine could directly stabilize the lysosome. As additional. evidence that this drug stabilizes membranes, we found that chlorphentermine inhibited the lysis of red blood cells in hypotonic solution. Acknowledoements--This research was supported by a Research Starter Grant from the Pharmaceutical Manufacturers Association Foundation, by the West Virginia Medical Corporation and by Biomedical Research Support Grant 5 S01 RR05433. The authors thank Warner Chilcott for their generous gift of chlorphentermine. The technical assistance of Ms Lynn H. Yenko and Ms Roberta Koshut is gratefully acknowledged. This research was presented, in part, at the 1978 Society of Toxicology Meeting, San Francisco, CA.
REFERENCES ABRAHAM R., HENDY R. d~, GRASSO P. (1968) Formation of myeloid bodies in rat liver lysosomes after chloroquine treatment. Exp. Molee. Path. 9, 212-229. CONCrnE J. & HAY A. J. (1959) Mammalian glycosidases. 2. Properties of a-mannosidase and fl-galactosidase from rat epididymis. Biochem. J. 73, 327-334. DI FRANCESCOC. & BICKEL M. H. (1977) Membrane lipids as intracellular binders of chlorpromazine and related drugs. Chem.-biol. Interact. 16, 335-346. DODGSON K. S., SPENCER B. & THOMASJ. (1955) Studies on sulfatases. 9. The arylsulfatases of mammalian livers. Biochem. J. 59, 29-37. DRENCKHAHN D., KLEIN'E L. & Li3LLMANN-RAUCH R. (1976) Lysosomal alterations in cultured macrophages exposed to anorexigenic and psychotropic drugs. Lab. Invest. 35, 116-123. FRANKEN G., L[JLLMANN H. & SIEGRIEDT A. (1970) The occurrence of huge cells in pulmonary alveoli of rats treated by an anorexic drug. Arzneim.-Forsch. 20, 417. HARTREE E. (1972) Determination of protein: A modification of the Lowry method that gives a linear photometric response. Analyt. Biochem. 48, 422 427. HRtmAN Z., SLESERSA. & HOPKINS E. (1972) Drug-induced and naturally occurring myeloid bodies. Lab. Int,est. 27, 62-70. HURST D. J., GARDNER D. G. & COFFIN D. L. (1970) Effect of ozone on acid hydrolases of the pulmonary alveolar macrophage. J. Reticulo. Soc. 8, 288-300. KARABELN1KD., ZBINDEN G. & BAUMGARTNERE. (1974) Drug-induced foam cell reaction in rats. I. Histopathologic and cytochemical observations with chlorphentermine, RMI 10.393 and Ro 4-4318. Toxic appl. Pharmac. 27, 395-407. LEVY G. A. & MCALLANA. (1963) Mammalian fucosidases. Biochem. J. 87, 361-367. LOCKARD V. G. & KENNEDY R. E. (1976) Alterations in rabbit alveolar macrophages as a result of traumatic shock. Lab. Invest. 35, 5OI-506. LL;LLMANN H., L[JLLMANN-RAUCHR. & WASSERMANNO. (1975) Drug-induced phospholipidosis. C R C Crit. Rev. Toxic. 2, 185-218. LOLLMANN-RAUCH R., REIL G.-H., ROSEN E. & SELLER K. U. (1972) The ultrastructure of rat lung changes induced by an anorectic drug (chlorphentermine). Virchows Arch. B. Zellputh. I1, 167-181.
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LOI.tMANN-RAucH, R. & REIL G.-[I. (1974) Chh:,rphentermine-induced lipidosis like ultrastructural alterations in lungs and adrenal glands of several species. Toxic appl. Pharmac. 30, 408-421. I..i-'IiMANN-RAucH R. (1974) Lipidosis-like ultrastructural alterations in rat lymph nodes after treatment with antidepressants or neuroleptics. Naunyn-Schmiedeherg.~. Arch. exp. Path. Pharmak. 286, 165 179. MERKOW L. P., EPSTEIN S. M.. SIDRANSKY H.. Vt-RNEY E. & PARt×) M. (1971) The pathogenesis of pulmonary aspergillosis. Ant. J. Path. 62, 57-74. PINe C. J. & L o w F. N. (1975) Scanning electron microscopy of cardiac endothelium of the dog. Am. ,l. Anat. 142. 137 158. Rl'ASOR M. J., TRUSII M. A. & WALKER E. R. (1978a) Changes in lysosomal properties of alveolar macrophages of rats treated with chlorphcntermine. Toxic appl. Pharrnac. In press. REASOR M. J., TRUSH M. A., ALDEN C. A. & WALKER E. R. (1978b) Characteristics of alveolar macrophage-derived giant cells resulting from chlorphentermine-induced phospholipidosis in the rat. Pharmacologist 20, 278. SELLINGER O. z., BEAUFAY H.. JAQUES p., DOYAN A. ~¢.
DE Duvl: C. (1960) Tissue fractionation studies. Intr;t cellvlar distribution and properties of f~-N-acet~,l glucosaminidase and fl'-galactosidase in rat liver. Bio¢'hcm../ 74, 450 456. SEYDN J K+ & WASSERMANN O. (1976) NMR-studies on the basis of drug-induced phospholipidosis-ll. Interaction between several amphiphilic drugs and phospholipids. Biochem. Pharmac. 25, 2357 2364. SNEDEC'OR G. W. & COCHRAN G. (1967) Statistical Mcthods 6th edn. Chap. 4, lov, a State University Press. Ames. Iowa. STAUBLI W., SCHWEIZER W. & SLITTER J. 119781 Some properties of myeloid bodies induced in rat Ii',er by an antidepressant drug (Maprotiline). Exp..'vtoh.c. Path. 278, 177-195. SVENDSON O. (1977)The effect of phenobarbital on chlorphentermine-induced alveolar foam cells in rats. 7~xi~ appl. Pharrnac. 40, 171. 173. TcR~m:t.L J. M. & NEtL M. W. (1969) The osmotic stability of lysosomes from adult and foetal guinea-pig livcr tissue. Biochem. J. I I I, 503-507. WEISSMANN G. (1966) Lysosomes and joint diseases. Arthritis Rheumat. 9, 834-839.
0306-3623 79 (13111-0083Stl2(~)0
L Y S O S O M A L STABILIZING E F F E C T OF C H L O R P H E N T E R M I N E IN THE RABBIT ALVEOLAR M A C R O P H A G E MARK J. REASOR and ELIZABETH R. WALKER Department of Pharmacology and Department of Anatomy, West Virginia University Medical Center, Morgantown, WV 26506, U.S.A. (Receit'ed 13 July 1978)
Abstract--l. Rabbits were injected (i.p.I daily with chlorphentermine 130mg;'kg), 5 days a week for 7 weeks. 2. Alveolar macrophages were collected from the lungs of control and treated rabbits by pulmonary lavage. 3. When compared to controls, the latencies of the lysosomal acid hydrolases, acid phosphatase and fl-N-acetyl glucosaminidasc were increased in lysosome-containing fractions from alveolar macrophages of treated rabbits. The same results were obtained when chlorphentermine was incubated in vitro with a lysosomal fraction from macrophages of normal rabbits. 4. The increased latency of lysosomal enzymes following chlorphentermine treatment can be explained by the drug's ability to stabilize lysosomal membranes.
in 0.154 M NaCI 5 days a week for 7 weeks. Control rabbits received an equal volume of vehicle. Because chlorphentermine is an anorectic drug, treated rabbits initially lost weight, recovered and proceeded to gain weight although at a rate less than non-injected controls. Therefore in this study, control rabbits were pair-fed to ensure weight gains comparable to the treated animals. One control and one treated animal were sacrificed on the same day, macrophages collected and the experiments performed.
INTRODI.'CT1ON The administration of a large number of amphiphilic drugs leads to a drug-induced phospholipidosis in many organs of experimental animals (LiJllmann et al., 1975). The disorder manifests itself within the lysosome of the cell resulting in the formation of lamellated lipid inclusions which have been termed "myeIoid bodies" (Abraham et al., 1968: Hruban et al., 1972; LiJllmann et al., 1975; Drenckhahn et al., 1976: St~iubli et al., 1978). Although these ultrastructural alterations have been described extensively, very little information exists as to specific effects on lysosomes which might lead to functional alterations in this organelle. St~iubli et al. (1978), have reported that treatment of rats with the amphiphilic drug maprotiline results in a labilization of hepatic lysosomes. We have administered chlorphentermine, another amphiphilic drug used commonly to induce this disorder. to rats, and observed changes in lysosomal properties of alveolar macrophages (Reasor et al., 1978a). In addition to resulting in an increase in activity of lysosomal acid hydrolases, treatment with chlorphentermine resulted in alterations in the integrity of the lysosomes as measured by the latencies of two acid hydrolases, acid phosphatase (AP) and fl-N-acetyl glucosaminidase (flNAG). The latency of AP increased while that of flNAG decreased. In an effort to extend our studies to other species, we examined the effects of chlorphentermine treatment on lysosomal properties of the alveolar macrophage of the rabbit, an animal in which drug-induced phospholipidosis has not been well documented. This report presents the results of this investigation. MA'I'ERIAI^S A N D
Collection of alveolar macrophages
Some time after the last drug administration (24 hr). the rabbits were deeply anesthetized with pentobarbital (50 mg/kg). The abdomen was opened by a midline incision and the animals exsanguinated by severing the abdominal aorta. The thorax was opened along the sternum and the lungs and trachea were removed intact. They were rinsed liberally with cold 0.154M NaCI and trimmed of extraneous tissue. The excised lungs were lavaged via the trachea with cold calcium and magnesium-free Hank's Balanced Salt Solution (HBSS), pH 7.4. Approximately 40ml were needed to fill the lungs, and the fluid was allowed to drain out by inverting the lungs. Approximately 250ml of fluid was collected, beyond which virtually no additional cells were recovered. Macrophages were recovered from the lavage fluid by sedimentation at 5000 for 10rain, washed twice and resuspended in complete H BSS.
METHODS
Animals and dru(.l administration
Chlorphentermine hydrochloride (Warner Chilcott, Morris Plains. N J) was administered to adult, male New Zealand white rabbits (2-3 kg) at a dose of 30mg/kg, i.p.. 83
Assays Protein content of the cells was measured according to the procedure described by Hartree (1972) using bovine serum albumin as standard. The following enzyme assays were performed on macrophages in the presence of 0.10,. Triton X-100:acid phosphatase -with p-nitrophenyl phosphate as substrate (Turnbull & Nell, 1969); /~-glucuronidase- with p-nitrophenyl-fl-o-glucuronide as substrate (Lockard & Kennedy. 1976): aryl sulfatase- with nitrocatechol sulfate as substrate (Dodgson et al.. 1955); flgalactosidase--with p-nitrophenyl-fl-D-galactoside as substrate (Levy & McAIlan, 1963), fl-N-acetyl glucosaminidase --with p-nitrophenyl-N-acetyl-fl-D-glucosaminide as substrate (Sellinger et al.. 1960): and a-mannosidase--with
84
M-XRK .I. RI',SOR and EII:Z.~BITII R. W.'~IK|R
p - n l t r o p h e n } ]--/-i)-nlanllosld¢ a~, '-;ubMrale I( "Ollchl¢ & H a } . 1959)
.'~lcasurement o! httencv As a measure of I.,,sosomal menlbrane stability, the latenties of .~,1~ and /~NA(-I ,acre determined. A bsosome-conraining fraction llS.0(~)g pellet) was prepared from homogeni/ed cells IHurst cta/.. 19701 and suspended in 11.25 M sucrose-20 m M Tris-acetate. ptl 7.4. All aliquot of this susp e n s , m 10.9 nail was added to I.I ml of complete HBSS at 37 (" and an aliquot removed immediately to determine baseline hydrolase release. The percent of enzyme activity released at this "'zero time" was measured by centrifuging a portion of tile aliquot sit 15.0(~) g h~r 20 mill and comparing tile non-sedimcntable activity with the total enzyme activity in the aliquot. The remainder of the sample was incubated [or 15ram at 37 (" and the percent of the enz3me acllviL', released into the non-sedimentabie form during this lime was determined as described. The latenc.~ of the enzyme activity was determined b', comparing the percent of the total ac~vit.~ released in 15rain with the baseline release. Macrophages were processed for electron microscopy as described b~ Pine & Lov, [1975). Red hh,od cell (RBC) lysts Blood was collected from tile central ear artery of rabbits b~ drainage through a 20 gauge needle into a heparinized tube. RBCs ~ere sedimented at 1000g for IOmin. washed twice and resuspended ill a medium containing 5 m M KCI. 5 m M glucose. 5raM III=.PES buffer, and 0.154M NaCI. pH 7.4. An equal vohmle of this medium containing chlorphcntcrmme Idouble the assay concentration) and xar.',ing concentrations of NaCI was added to the RBC suspension to gi~e the desired drug concentration and osrnolarit',. Incubation was carried out for 15min at 3"7,(" An aliquot `aas then remo,,ed and the RB('s sedimented at 10Ollq for 1 0 r a m The supernatant phase ',',,is rem
W h e n c o m p a r e d to controls, a slight but nonsignificant increase was o b s e r v e d in the n u m b e r of m a c r o -
Table I. Effect of chlorphentermine adminislration on the recovery of alveolar macrophages ['rolT1 rabbit lung,, b', pulmonary la~age
Macrophages
("ells kg b.`a. I× 10 I
mkz Protein Ill- Ceils
('ontrol
2.67 4- 11.61"
1.80 ± (I. It)+
Treated
3.61 ± 0.52
2.(12 + 024
Rabbits i.p.I for 7 * Mean .t- Mean
were treated ~,ith chlorphcntermine 130nlg kg weeks. 5 da~s per week. 4: S.t-.M. [N - 71. Jr S.E.M. IN = 4i.
p h a g e s recovered from the l u n g s of treated rabbits (Table 1). T h e p r o t e i n c o n t e n t per 10" cells was not significantly different either. Electron m i c r o g r a p h s ~not s h o w n ) o f alveolar m a c r o p h a g e s from the d r u g - t r e a t e d a n i m a l s failed to show the characteristic accumulation of lysosomallyderived lamellated inclusions w h i c h are a s s o c i a t e d with c h l o r p h e n t e r m i n e a d m i n i s t r a t i o n in o t h e r species ( L i J l l m a n n - R a u c h et al.. 1972: L i i l l m a n n - R a u c h & Rei[, 19741. T h e similarity of t h e u l t r a s t r u c t u r e of the m a c r o p h a g e s from the c o n t r o l a n d c h l o r p h e n t e r m i n e treated r a b b i t s indicated that s e v e n weeks of d r u g t r e a t m e n t h a d failed to i n d u c e p h o s p h o l i p i d o s i s as .judged by a m o r p h o l o g i c criterion. Since t h e l y s o s o m e is n o r m a l l y t h e first o r g a n e l l e visibly affected in this d i s o r d e r ( L i i l l m a n n et al., 19751 it was of interest to d e t e r m i n e w h e t h e r functional c h a n g e s h a d o c c u r r e d within t h e l y s o s o m e w i t h o u t the d e v e l o p m e n t o f t h e lamellated inclusions. T h e activities of six l y s o s o m a l acid h y d r o l a s e s were m e a s u r e d a n d f o u n d not to differ in the two g r o u p s w h e n e x p r e s s e d as a f u n c t i o n o f cell n u m b e r or cell p r o t e i n i T a b l e 2). L y s o s o m a l m e m b r a n e stability is a n i m p o r t a n t d e t e r m i n a n t of l y s o s o m a l f u n c t i o n w i t h i n t h e celL As a m e a s u r e of m e m b r a n e stability, t h e latencies of two l y s o s o m a l acid h y d r o l a s e s , A P a n d /~NAG were e x a m i n e d in alveolar m a c r o p h a g e s following chlorp h e n t c r m i n e t r e a t m e n t (Table 3). F o l l o w i n g 15 m i n of i n c u b a t i o n t h e r e was a 117", increase in t h e release of A P from a l y s o s o m e - c o n t a i n i n g fraction p r e p a r e d from the c o n t r o l m a c r o p h a g e s . In c o n t r a s t , t h e r e was only a 70",, increase in A P release from t h e prepa r a t i o n from cells of treated rabbits. T h i s c o m p a r i s o n indicated that t h e r e was a significant i n h i b i t i o n [P < 0.05) in the release o f A P (increased latency1 in s a m p l e s from the c h l o r p h e n t e r m i n e - l r e a t e d rabbits. A
Table 2. The effect of chlorphentermine administration on lysosomal acid hydrolase activities of rabbit alveolar macrophages
Macrophages
Acid phosphatase
Control Treated
141.0 z 26.5* 159.3 ~_ 34.6
21.9 4- 2.0 24.0 4- 4.3
Control Treated
81.4 ± 18.5 80.6 4- 14.7
12.4 _. 1.7 12.6 _.- 0.9
fl-(ilucuronidase
Aryl sulfatase
/1-Galactosidase
(nmoles.:min 10" cells) 15.5 4- 1.0 58.2 _+ 2.6 18.2 + 2.() 6(I.9 _+ 10.6 {nmoles.'min:mg protein) 8.7 4- 0.6 32.8 _~_ 2.9 9.1 _-2 0.7 29.7 -* 3.4
/'¢-N-Acetylglucosaminidase
:~-Mannosidase
857.8 _+ 82.7 977.6 +_ 141.1
38.4 4T 4.8 37.0 + 8.9
483.6 ± 61.0 481.3 _+ 26.7
21.8 t_ 3.5 17.9 _+ 2.8
Rabbits were treated with chlorphentermine {30mg'kg) for 7 weeks. 5 days per week. * Mean 4- S.E.M. IN = 4~.
85
Chlorphentermine and lysosomes Table 3. The effect of chlorphentermine administration on the latencies of lysosomal acid hydrolases of rabbit al~.eolar macrophages Macrophages
Time of incubation (min)
Acid phosphatase
/3-N-Acetylglucosaminidase
of Total Activity Released) 7.3 + 0.5* 28.4 + 5.7
(".
0 Control 15 0
15.7 + 1.2 45.7 + 5.3 (*117.0 _+ 21.1",,1 (T72.2 _+ 16.0",,) 8.4 _+ 0.6 30.8 _+ 4.2
Treated 15
14.2 _+ 0.9 (]'69.8 +_ 6.5",,)
mine on RBC stability in hypotonic solution was examined. At 10- 3 M, chlorphentermine inhibited the lysis of RBCs as demonstrated by the shift of the lysis curve to the left (Fig. 2). This plasma m e m b r a n e stabilization occurred when RBCs were incubated in media from 174 to 191 m O s M . The dose-response relationship of chlorphentermine and the inhibition of lysis at 191 m O s M is presented in Fig. 3 and demonstrates that under these conditions the drug is an effective inhibitor of RBC lysis, presumably by stabilizing the plasma m e m b r a n e of the cell. Therefore, in two separate systems in vitro, chlorphentermine is capable of stabilizing membranes.
43.7 _+ 3.9 (T45.3 _+ 9.6".)
Rabbits were treated with chlorphentermine 130mg'kg) for 7 weeks, 5 days per week. * Mean + S.E.M. (N = 4).
similar inhibition in the release of flNAG was observed in the same samples from treated animals. Therefore, while not leading to ultrastructural changes within the lysosome, these results indicated that the chronic administration of chlorphentermine had apparently resulted in an increased stabilization of the lysosomal membrane. To examine this further, the direct effect of chlorphentermine on lysosomal enzyme release was investigated (Fig. 1). The incubation of chlorphentermine with a lysosome-containing fraction in vitro led to an inhibition in the release of both A P and flNAG, particularly at the higher drug concentrations. Thus, chlorphentermine has the ability to directly stabilize lysosomal membranes, at least in vitro. To test this hypothesis about m e m b r a n e stabilization using another system, the effect of chlorphenter-
DISCL:SSION One of the most striking changes in rat, mouse, and guinea pig lung tissue following the daily adminstration (for 5 weeks or less) of chlorphentermine is the appearance of an increased number of free cells in the alveoli (Franken et al., 1970; Karabelnik et al., 1974; Liillmann-Rauch & Reil, 1974; Svendsen, 1977: Reasor et al., 1978b). These cells, which appear to be derived from alveolar macrophages, are enlarged and contain lamellated inclusions resulting in their being termed "foam cells" (Karabelnik et al., 1974; Svendsen, 1977). These inclusions have been shown to be lysosomal in origin (LiJllmann-Rauch & Reil, 1974; Liillman et al., 1975) and it is proposed that they arise as a result of the formation of a complex between the parent drug and phospholipids (Liillmann et al., 1975). This interaction prevents the normal lysosomal degradation of these lipids from occurring leading to their accumulation within the cell and the subsequent formation of the lamellated structures. In a study comparing the ultrastructural changes induced by chlorphentermine in several organs and
70 ~ ] 60
~c.d Phosphotose .~- I%- '~cetylqluCOSOm~nlOoSe
50
4O
T.
L.
3O 2O IO
0
10-3
10.4
5xlO"4
5xlO"5
CONC.(M)
Fig. 1. The effect of chlorphentermine on the release of acid hydrolases from lysosome-containing preparations from rabbit alvcolar macrophagcs. A lysosome-containing fraction was prepared from the alveolar macrophages of normal rabbits and the effect of various concentrations of chlorphenterminc on the release of acid phosphatase (clear bars) and fl-N-acetyl glucosaminidase (stippled barsl was determined (see Materials and Methods for detailed procedure). The values represent the percent inhibition of enzyme rclcase (Mean +. SE.M., N = 5) when compared to fractions incubated in the absence of drug. *P < 0.05.
86
MARK J. RLASOR a n d ELIZABI i n R. V~.:AIK I R
I00
80
60
40
20
A
i
~
120
140
160
i
180
"(>'--i- -
0
200
-
~
220
240
260
mOsM
Fig, 2. The effect of chlorphentermine on the lysis of rabbit red blood cells in hypotonic solution. Rabbit red blood cells were incubated for 15 min, 37 (', in media of varying osmolarity and the percent lysis was measured in the absence ( • ) and presence of chlorphentermine II0 ~ M~ t O ). Each point represents the mean of two experiments. See Materials and Methods for media composition and procedure for measurement of lysis, species. Liillmann-Rauch & Reil (1974) reported that after 12- 14 weeks of drug administration to rabbits (at levels comparable to that used in the present study), only a slight increase in the numbers of macrophages were seen in the alveoli. However, foam cells were present at this time. The present study revealed that no increase in cell number, cell size [as measured by protein content, a parameter which correlates with increased cell size in rat alveolar foam cells (Reasor et al.. 1978b and unpublished results)] or cells of a b n o r m a l morphology appeared in the alveoli of rabbits treated daily with chlorphentermine for 7 weeks. The basis for the difference in susceptibility between the rabbit and other species is unknown. One possibility would be differences in the pulmonary uptake and.'or metabolism of chlorphentermine between these species. This notion is speculative at present,
and a more delinitive explanation awaits research in this area. Even though chlorphentermine administration did not result in the characteristic ultrastructural changes in rabbit alveolar macrophages, specific effects on lysosomal properties resulted. A stabilization of the lysosomal m e m b r a n e appeared to occur in these ceils, and evidence was obtained indicating that the drug may exert this effect by interaction with the membrane. Seydel & W a s s e r m a n n (1976) have shown that chlorphentermine binds to phospholipids, thus providing a molecular basis for this hypothesis. The ability to interact with m e m b r a n e s may be a general characteristic of amphiphilic drugs. Chlorpromazine, an amphiphilic drug reported to induce phospholipidosis (Liillmann-Rauch. 1974) binds to membranes (DiFrancesco & Bickel, 1977i with the major
80
T,
U3
I
> J
60
o z
o i-
40
z
2O
10-3
10-4
10-5
CHLORPHENTERMINE
10-6 (M)
Fig. 3. The dose-dependent inhibition of lhe Ivsis of rabbit red blood cells by chlorphentermine. Rabbit red blood cells were incubated for 15min, 37 ('+ in media of 191 mOsM with ",aoing concentrations of chlorphentermine, and the inhibition of lysis was measured and compared to samples incubated in the absence of drug. Values ;ire the Mean _+ S.E.M. with IN) shown at the base of each bar. See Materials and Methods for media composition and procedure fl~r measuremenl oflysis.
Chlorphentermine and lysosomes site being the non-polar moieties of phospholipids. Chloroquine, which also causes phospholipidosis (Abraham et al., 1968; Liillman et al., 1975) has been shown to stabilize lysosomal membranes (Weissmann, 1966) in vivo and in vitro. Rather high levels of chlorphentermine were required to stabilize lysosomes in vitro in the present study, and it can be questioned as to whether such levels would be reached in vivo with this dosing regimen. Since the concentration of drug was not measured in the macrophage, or more specifically in the lysosomal membrane, this information is not available. In fact, to the best of our knowledge, such measurements have never been reported for lipidosisproducing drugs. Liillmann et al. (1975) speculate that since high levels of drugs are usually administered for long periods of times in such studies, concentrations up to 1 0 - 3 M may be reached in certain organs. If the drugs preferentially bind to phospholipids within membranes as shown for chlorpromazine (DiFrancesco & Bickel, 1977), their actual concentrations at these sites may, in fact, be even higher. The functional implications of lysosomal stabilization in the alveolar macrophage must be considered. Corticosteroid administration leads to an increased stability of macropbage lysosomes and consequently to an increased susceptibility to pulmonary aspergillosis in mice (Merkow et al., 1971). Therefore, treatment with other drugs with similar actions may have serious consequences for the host organism if this leads to an impairment in pulmonary defense. The administration of amphiphilic drugs does have physiological significance in vivo as is evidenced by the work presented by Liillman et al. (1975). They found that the formation of paw edema induced by dextran in rats was inhibited by the chronic pretreatment with amphiphilic drugs. Since the lysosomal system participates in this inflammatory disorder, one explanation of the results is that drug administration led to an inhibition of lysosomal enzyme activity. This seems unlikely since no effect was found on the activities of six lysosomal enzymes in the present study. Another, and one consistent with the results presented here. is that the drugs stabilized the lysosomes reducing their involvement in the response. Perhaps the most significant result of this study is that an amphiphilic drug can have effects on iysosomal properties of alveolar macrophages without inducing the formation of lamellated inclusions. Therefore, in addition to ultrastructural changes, additional parameters should be investigated when assessing the effects of amphiphilic drug administration. CONCLUSION The increased latencies of lysosomal enzymes in rabbit alveolar macrophages following chronic treatment with chlorphentermine can be explained by the drug's ability to stabilize lysosomal membranes. SUMMARY
The chronic administration of chlorphentermine to rabbits failed to induce phospholipidosis in alveolar macrophages as judged by the absence of ultrastructural changes within the lysosome of the cell. How~.,.P. 10.'2 a
87
ever, a specific effect on the lysosome was observed following drug-treatment. An increased stabilization of the lysosome resulted as measured by an increase in the latencies of the lysosomal acid hydrolases, acid phosphatase and fl-N-acetyl glucosaminidase. The addition of chlorphentermine to a lysosome-containing fraction led to an increase in the latencies of these two hydrolases, indicating that chlorphentermine could directly stabilize the lysosome. As additional. evidence that this drug stabilizes membranes, we found that chlorphentermine inhibited the lysis of red blood cells in hypotonic solution. Acknowledoements--This research was supported by a Research Starter Grant from the Pharmaceutical Manufacturers Association Foundation, by the West Virginia Medical Corporation and by Biomedical Research Support Grant 5 S01 RR05433. The authors thank Warner Chilcott for their generous gift of chlorphentermine. The technical assistance of Ms Lynn H. Yenko and Ms Roberta Koshut is gratefully acknowledged. This research was presented, in part, at the 1978 Society of Toxicology Meeting, San Francisco, CA.
REFERENCES ABRAHAM R., HENDY R. d~, GRASSO P. (1968) Formation of myeloid bodies in rat liver lysosomes after chloroquine treatment. Exp. Molee. Path. 9, 212-229. CONCrnE J. & HAY A. J. (1959) Mammalian glycosidases. 2. Properties of a-mannosidase and fl-galactosidase from rat epididymis. Biochem. J. 73, 327-334. DI FRANCESCOC. & BICKEL M. H. (1977) Membrane lipids as intracellular binders of chlorpromazine and related drugs. Chem.-biol. Interact. 16, 335-346. DODGSON K. S., SPENCER B. & THOMASJ. (1955) Studies on sulfatases. 9. The arylsulfatases of mammalian livers. Biochem. J. 59, 29-37. DRENCKHAHN D., KLEIN'E L. & Li3LLMANN-RAUCH R. (1976) Lysosomal alterations in cultured macrophages exposed to anorexigenic and psychotropic drugs. Lab. Invest. 35, 116-123. FRANKEN G., L[JLLMANN H. & SIEGRIEDT A. (1970) The occurrence of huge cells in pulmonary alveoli of rats treated by an anorexic drug. Arzneim.-Forsch. 20, 417. HARTREE E. (1972) Determination of protein: A modification of the Lowry method that gives a linear photometric response. Analyt. Biochem. 48, 422 427. HRtmAN Z., SLESERSA. & HOPKINS E. (1972) Drug-induced and naturally occurring myeloid bodies. Lab. Int,est. 27, 62-70. HURST D. J., GARDNER D. G. & COFFIN D. L. (1970) Effect of ozone on acid hydrolases of the pulmonary alveolar macrophage. J. Reticulo. Soc. 8, 288-300. KARABELN1KD., ZBINDEN G. & BAUMGARTNERE. (1974) Drug-induced foam cell reaction in rats. I. Histopathologic and cytochemical observations with chlorphentermine, RMI 10.393 and Ro 4-4318. Toxic appl. Pharmac. 27, 395-407. LEVY G. A. & MCALLANA. (1963) Mammalian fucosidases. Biochem. J. 87, 361-367. LOCKARD V. G. & KENNEDY R. E. (1976) Alterations in rabbit alveolar macrophages as a result of traumatic shock. Lab. Invest. 35, 5OI-506. LL;LLMANN H., L[JLLMANN-RAUCHR. & WASSERMANNO. (1975) Drug-induced phospholipidosis. C R C Crit. Rev. Toxic. 2, 185-218. LOLLMANN-RAUCH R., REIL G.-H., ROSEN E. & SELLER K. U. (1972) The ultrastructure of rat lung changes induced by an anorectic drug (chlorphentermine). Virchows Arch. B. Zellputh. I1, 167-181.
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MARK J. REASOR and ~LIZAB|-.II{ R. WAI,KER
LOI.tMANN-RAucH, R. & REIL G.-[I. (1974) Chh:,rphentermine-induced lipidosis like ultrastructural alterations in lungs and adrenal glands of several species. Toxic appl. Pharmac. 30, 408-421. I..i-'IiMANN-RAucH R. (1974) Lipidosis-like ultrastructural alterations in rat lymph nodes after treatment with antidepressants or neuroleptics. Naunyn-Schmiedeherg.~. Arch. exp. Path. Pharmak. 286, 165 179. MERKOW L. P., EPSTEIN S. M.. SIDRANSKY H.. Vt-RNEY E. & PARt×) M. (1971) The pathogenesis of pulmonary aspergillosis. Ant. J. Path. 62, 57-74. PINe C. J. & L o w F. N. (1975) Scanning electron microscopy of cardiac endothelium of the dog. Am. ,l. Anat. 142. 137 158. Rl'ASOR M. J., TRUSII M. A. & WALKER E. R. (1978a) Changes in lysosomal properties of alveolar macrophages of rats treated with chlorphcntermine. Toxic appl. Pharrnac. In press. REASOR M. J., TRUSH M. A., ALDEN C. A. & WALKER E. R. (1978b) Characteristics of alveolar macrophage-derived giant cells resulting from chlorphentermine-induced phospholipidosis in the rat. Pharmacologist 20, 278. SELLINGER O. z., BEAUFAY H.. JAQUES p., DOYAN A. ~¢.
DE Duvl: C. (1960) Tissue fractionation studies. Intr;t cellvlar distribution and properties of f~-N-acet~,l glucosaminidase and fl'-galactosidase in rat liver. Bio¢'hcm../ 74, 450 456. SEYDN J K+ & WASSERMANN O. (1976) NMR-studies on the basis of drug-induced phospholipidosis-ll. Interaction between several amphiphilic drugs and phospholipids. Biochem. Pharmac. 25, 2357 2364. SNEDEC'OR G. W. & COCHRAN G. (1967) Statistical Mcthods 6th edn. Chap. 4, lov, a State University Press. Ames. Iowa. STAUBLI W., SCHWEIZER W. & SLITTER J. 119781 Some properties of myeloid bodies induced in rat Ii',er by an antidepressant drug (Maprotiline). Exp..'vtoh.c. Path. 278, 177-195. SVENDSON O. (1977)The effect of phenobarbital on chlorphentermine-induced alveolar foam cells in rats. 7~xi~ appl. Pharrnac. 40, 171. 173. TcR~m:t.L J. M. & NEtL M. W. (1969) The osmotic stability of lysosomes from adult and foetal guinea-pig livcr tissue. Biochem. J. I I I, 503-507. WEISSMANN G. (1966) Lysosomes and joint diseases. Arthritis Rheumat. 9, 834-839.