Download In Vitro and in Vivo Ultrastructural Changes Induced by Macrolide

FUNDAMENTAL AND APPLIED TOXICOLOGY 32, 2 0 5 - 2 1 6 (1996)
AjmCLENO. 0123
In Vitro and in Vivo Ultrastructural Changes Induced
by Macrolide Antibiotic LY281389
J. W. HORN, C. B. JENSEN, S. L. WHITE, D. A. LASKA, M. N. NOVILLA, D. D. GIERA, AND D. M. HOOVER
Toxicology Research Laboratories, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, Indiana
46140-2517
Received August 31, 1995; accepted April 26, 1996
al., 1989, 1990), the antihistaminic agents chlorcyclizine
In Vitro and in Vivo Ultrastructural Changes Induced by Mac- (Hruban et al., 1972) and meclazine (Reasor, 1989), and
rolide Antibiotic LY281389. HORN, J. W., JENSEN, C. B., WHITE,
S. L., LASKA, D. A., NOVILLA, M. N., GIERA, D. D., AND HOOVER,
D. M. (1996). Fundam. AppL Toxicol. 32, 205-216.
High doses of LY281389 (9-N-(n-propyl)-erythromycylamine) cause cytoplasmic vacuolar changes in striated and smooth
muscle characteristic of drug-induced phospholipidosis. This
study characterized phospholipidosis in striated and smooth
muscle of rats and dogs, compared in vivo observations with
those in a cultured rat myoblast model, and attempted to confirm the lysosomal origin of the drug-induced vacuoles. Standard transmission electron microscopy and acid phosphatase
cytochemistry techniques were used to evaluate ultrastructural
changes in vivo and in vitro. Rats and dogs exposed to
LY281389 had a time- and dose-related increase in number
and size of vacuoles containing concentric lamellar figures in
cardiac and skeletal muscle. Cytochemical staining of dog
stomach smooth muscle for acid phosphatase, a lysosomal enzyme, stained the periphery of vacuoles that contained concentric lamellar figures. Cultured rat L6 myoblast cells were exposed to 0.25 mg LY281389/ml for 2.5, 5, 10, 20, 30, or 90 min
and 2, 6, 12, 24, or 48 hr. Cell cultures exposed for 2 hr had
several predominantly large, clear, membrane-bound vacuoles,
and at 6 and 12 hr there were greater numbers of large vacuoles
that contained increased amounts of membranous figures. Following 24- or 48-hr exposures, vacuoles occupied most of the
cytoplasmic volume, and were engorged predominantly with
amorphous or granular material. These findings indicate that
LY281389 can induce similar phospholipidosis-like vacuolar
changes in rat and dog muscle and in a cultured rat muscle
cell line. Further, positive acid phosphatase staining of druginduced vacuolar structures, in conjunction with standard
transmission electron microscopy techniques, strongly suggests
that vacuoles seen in vitro and in vivo are lysosomal in origin.
C 1996 Sodety of Toxicology
More than 30 drugs with a variety of pharmacological
indications are known to induce phospholipidosis in humans, animals, or cell culture (Ltillmann-Rauch, 1979).
Drugs such as the antibiotics cephaloridine (Laska et al.,
1990), streptomycin (Sens et al., 1991), and gentamicin
(Reasor, 1989), the antiarrhythmic drugs amiodarone
(Joshi and Mehendale, 1989) and disobutamide (Ruben et
205
the antimalarial chloroquine (Lullmann-Rauch, 1979;
Hruban et al., 1972; Hostetler et al., 1985) have been
reported to induce phospholipidosis, myeloid bodies, or
intralysosomal storage of polar lipids. These and other
phospholipidosis-inducing compounds have similarities
in their chemical structures, i.e., a hydrophobic component consisting of an aromatic ring structure and a hydrophilic region containing a primary or substituted amino
group uncharged at physiological pH, but charged at low
pH (Lullman»-Rauch, 1979; Reasor, 1989). These compounds are commonly referred to as cationic amphiphilic
drugs (CADs).
By virtue of their chemical properties, CADs readily
interact with membrane phospholipids, and can be accumulated in lysosomes by endocytosis (Hjelle and Ruben,
1989; Ruben et al., 1989) or by passively permeating
cellular and subcellular membranes (Lilllmann-Rauch,
1979). Several possible mechanisms by which drugphospholipid interactions can interfere with phospholipid
metabolism have been proposed (Hostetler, 1984; Joshi
and Mehendale, 1989; Ltillmann-Rauch, 1979; Reasor,
1989). Current theories suggest that lysoj-omal phospholipase activities may be inhibited by the unavailability of
the phospholipid substrate due to drug binding; by the
direct binding of drug to the phospholipase enzyme(s);
or by inactivation of lysosomal enzymes due to increases
in lysosomal pH. At present, insufficient information is
available to conclude that any one mechanism is responsible for drug-induced phospholipidosis.
CAD-induced phospholipidosis occurring in vitro and
in vivo is characterized morphologically by distinct cytoplasmic membrane-bound vacuoles that contain varying
amounts of concentric lamellar membranous figures (Ulrich etal., 1991), clear contents (Hjelle and Ruben, 1989;
Ruben et al., 1990), or amorphous material (Hruban et
al., 1972; Lullmann etal., 1978; LUllmann-Rauch, 1979;
Reasor, 1984; Ruben et al., 1989). Much attention has
been focused on the relationship between lysosomal drug
accumulation, phosphulipidosis, and toxicity. High doses
or prolonged exposures to some phospoholipiddtic drugs
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HORN ET AL.
,i
1
FIG. 1. Transmission electron micrograph of rat cardiac myocytes following 1 month of exposure to 300 mg LY281389/kg/day. Densely stained
concentric lamellar figures (arrows) are in contact with mitochondna between myofibnls. Bar = 1 \im.
\.
FIG. 2. Transmission electron micrograph of rat quadriceps femoris muscle following I month of exposure to 300 mg LY281389/kg/day. Most
vacuoles contain electron-dense concentric lamellar figures (arrows), or some had dense amorphous material (arrowheads). Bar = I ^m.
207
LY281389-INDUCED ULTRASTRUCTURAL CHANGES
: *
r
FIG. 3. Transmission electron micrograph of dog stomach smooth muscle following a 7-day exposure to 100 mg LY281389/kg. Membrane-bound
lamellar figures are located in close proximity to nuclei and at the perimeter of the cell (arrows). Bar = 1 /jm.
\
ft
FIG. 4. Transmission electron micrograph of dog stomach smooth muscle following a 14-day exposure to 100 mg LY281389/kg. Longer compound
exposures increase the amount of membrane-bound lamellar figures (arrowheads). Bar = 1 fim.
208
HORN ET AL.
TABLE 1
In Situ Cellular Viability Results: Fluorometric Analysis
Concentration
(mg LY281389/ml)
0
0.01
0.05
0.10
0.25
0.50
1.00
BCECF-AM'
(relative fluorometric units)
3660
3613
3318
2989
3056
2054
591
Uptake
(% of control)
± 84*
± 39
± 18
± 71
± 82
± 297
±
5
100
99
91
82
84
56
16
° Cell viability was measured using 2,7-bis(2-cartx>xyethyl)-5,6-carboxyfluorexcein-acetoxymethyl ester (BCECF-AM), a vital fluorescent dye.
* Mean ± SE.
may lead to dose-limiting toxicities, including nephrotoxicity with gentamicin (Appel and Neu, 1977), pulmonary
toxicity with amiodarone (Rotmensch et al., 1980), and
myopathy and retinopathy with chloroquine (Hughes et
al., 1971; Hobbs et al., 1959). Although the mechanisms
of these toxicities are not fully understood, recent findings
suggest that drug accumulation may cause functional impairment of lysosomes (Gladue et al., 1989; Martin et
al., 1985), with subsequent disruption of phospholipid
metabolism and, in some cases, cell death (Lilllmann et
al., 1978; Kaloyanides and Pastoriza-Munoz, 1980).
In a toxicological evaluation of the experimental macrolide antibiotic LY281389 in the dog, high tissue levels
of the drug were observed, vacuolation was found in a
variety of tissues, and skeletal muscle toxicity was the
primary target organ toxicity (Roesner et al., 1991). The
purpose of the present work was to investigate sequential
cellular changes leading to vacuolization following exposure to LY281389, and to attempt to determine the mor-
phogenesis of these vacuoles. Additionally, we compared
the in vivo changes with those in an in vitro muscle cell
line to determine whether the in vitro cellular system
could be predictive of in vivo cytologic changes and, potentially, muscle toxicity. Finally, selected tissues and
cells were cytochemically stained for acid phosphatase
(AP) to determine whether drug-induced vacuolar structures were derived from lysosomes.
MATERIALS AND METHODS
Animal exposures. Experiments were conducted with beagle dogs
to investigate subchronic and subacute effects of LY281389 exposure.
Young adult beagle dogs were given 0, 25, or 75 mg LY281389/kg/day
in the diet for 30 days. At the end of dosing one group of animals
was necropsied and one group of animals was selected for a 30-day
reversibility phase and necropsied at the termination of this phase. Additionally, in a subacute study, beagle dogs were given 100 mg LY281389/
kg/day in the diet for 0, 2, 7, or 14 days. Twenty-four hours after the
last dose all animals were necropsied and stomach smooth muscle, heart,
and quadriceps femoris muscle were prepared for transmission electron
microscopy (TEM) examination. Acid phosphatase cytochemistry for
staining of lysosomes was performed on smooth muscle from stomachs
of selected dogs
Fischer 344 rats were given 0 or 300 mg LY281389/kg/day by gavage
for 30 days. Samples of heart and skeletal muscle (quadriceps femoris)
were collected from rats at necropsy 24 hr after the last gavage dose for
TEM examination.
Tissue culture. L6 cells [CRL 1458, American Type Culture Collection
(ATCC), Roclcville, MD], a rat skeletal muscle myoblast cell line, were
maintained with medium 199 (GIBCO, Grand Island, NY) supplemented
with 10% fetal bovine serum (GIBCO), and grown at 37°C in 5% CO2
atmosphere. Maintenance medium was replaced twice a week, and cultures
were passed weekly using 0.25% trypsin-0.53 nun EDTA (GIBCO). Cultures were maintained at a subconfluent density before study initiation to
preserve their myoblastic state, as suggested by the ATCC.
Pilot cytotoxicity experiments were performed to identify concentrations
of LY281389 that caused morphologic changes observable at the light
microscopic level, with minimal effect on cellular viability over the desired
time points. For these experiments, L6 cells were seeded in 24-well culture
plates at 3.0 x 105 cells/well in maintenance medium and incubated at 37"C
TABLE 2
In Vitro Results: Induced Lysosomal Effects in L6 Cultured Cells Treated with 0.25 mg/ml LY281389
Duration of
exposure
Control
2.5 min
5 min
10 min
20 min
30 min
90 min
2 hr
6hr
12 hr
24 hr
48 hr
Lysosome number/size
Rare to no lysosomes/small
Rare to no lysosomes/small
Rare to no lysosomes/small
Rare/small
Few/small
Several/medium
Several/medium to large
Several/medium to large
Many/large
Many/large
Numerous/large and variably sized
Numerous/large and variably sized
Lysosome content
Clear
Clear
Clear
Clear
Clear; rare small membrane figures
Rare amorphous granules; few clear; many membrane figures
Rare clear, many membrane figures
Predominantly membrane figures
Rare amorphous material; rare clear, numerous membrane figures
Few amorphous material; many membrane figures
Amorphous material
Amorphous material
LY281389-INDUCED ULTRASTRUCTURAL CHANGES
209
tion of 10 /ig/ml of BCECF-AM in SPRFM was incubated at 37°C for 40
min. The BCECF/SPRFM solution was removed by aspiration, cultures
were rinsed twice with SPRFM, and an additional amount of SPRFM was
placed over the monolayer. Fluorescence was expressed as a percentage of
the control fluorescence signal.
Acid phosphatase cytochemistry. Replicate L6 cell cultures, at all
time points except 2.5 min, and dog stomach smooth muscle were processed for AP staining. Tissues were fixed in modified Karnovsky's
fixative for 1 hr and rinsed in 0.1 M cacodylate buffer (pH 7.2) for 18
hr at 4°C. Representative samples of L6 cells and smooth muscle tissue
were rinsed in three 10-min exchanges of 0.05 M acetate buffer with
7% sucrose (pH 5) at room temperature, and incubated in 0.1 M acetatebuffered cytidine 5'-monophosphate-cerium chloride medium for a total of 90 min at 37°C, with fresh incubation medium applied at 30min intervals. Additional tissue samples were incubated with nutrient
medium neutralized with sodium fluoride to serve as processing controls. All samples were rinsed in three exchanges of 0.05 M acetate
buffer prior to secondary fixation in 2% buffered osmium tetroxide (pH
7.2). Processing for TEM consisted of dehydration with serially graded
ethanol solutions and embedment in an epoxy resin.
Transmission electron microscopy processing. Representative samples of heart and skeletal muscle (quadriceps femoris) of rats and dogs
A
FIG. 5. Transmission electron micrograph of control L6 muscle cells.
Note constituent cellular organelles. G, Golgi; solid arrows, mitochondria;
N, nuclei; arrowheads, rough endoplasmic reticulum; hollow arrows, support membrane. Bar = 1 /xm.
for 48 hr with 0, 0.01, 0.05, 0.10, 0.25, 0.50, or 1.0 mg LY281389/ml.
From these pilot studies, the 0.25 mg LY281389/ml dose group revealed
morphologic changes desirable for qualitative and semiquantitative assessment
L6 cells were seeded at 2.0 x 10* cells/dish in polycarbonate filterlined 35-mm tissue culture dishes in maintenance medium that yielded a
monolayer on attachment. The following day, the growth medium was
removed and cultures were rinsed with fresh medium without serum. Cultures were incubated for 2.5, 5, 10, 20, 30, or 90 min, or 2, 6, 12, 24, or
48 hr at 37°C in medium containing 0.25 mg LY281389/ml. This procedure
was performed in duplicate for each time point.
Viability. Cell viability was measured using 2,7-bis(2-carboxyethyl)5, 6-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) (Molecular
Probes, Eugene, OR), a nonpolar molecule freely traversing the cellular
membrane and cleaved by nonspecific cytosolic esterases. Once cleaved,
the probe is trapped in viable cells and can be quanititated fluorometrically.
Fluorescence was measured in situ using a Cytofluor plate reader (Millipore,
Bedford, MA) at 485-nm excitation and 530-nm emission wavelengths.
Following the 48-hr LY281389 incubation, cultures were rinsed twice
with serum-free RPMI 1640 medium without phenol red (SPRFM). A solu-
FIG. 6. Transmission electron micrograph of L6 muscle cells exposed
to 0.25 mg LY281389/ml for 30 min. Representative L6 cells have increased
vacuolation containing small membranous figures (arrowheads). Bar = 1
/*m.
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HORN ET AL.
S
FIG. 7. Transmission electron micrograph of L6 cells exposed to 0 25 mg LY281389/ml for 6 hr. At this time of exposure, amount and size of
vacuolation have increased and the contents are predominantly membranous figures (arrowheads) with some rare amorphous matenal (arrows). Bar
= 1 fim.
and stomach smooth muscle of dog and polycarbonate filter-supported
cell cultures were fixed overnight in modified Karnovsky's fixative at
4°C; buffer rinsed with 0.1 M cacodylate buffer (pH 7.2); and secondarily
fixed with 2% buffered osmium tetroxide (pH 7.2) for 1 hr. After three
rinses in 0.1 M cacodylate buffer, all tissues were dehydrated in serially
increasing concentrations of ethanol solutions, infiltrated for 2 hr with
an equal mixture of either Poly/Bed 812 resin (Polysciences, Inc , Warnngton, PA) and propylene oxide (animal tissues) or Poly/Bed 812 resin
and 100% ethanol (cell cultures), and embedded with 100% Poly/Bed
812 epoxy resin. Polymerization was complete after 24 hr at 37°C and
48 hr at 60°C.
Ultrathin sections were cut with a diamond knife, mounted on either
copper 50-mesh Formvar-coated grids or uncoated 200-mesh copper
grids, and counterstained with uranyl acetate and lead citrate. Ultrathin
sections were evaluated with a Philips 4I0LS (Philips Electronic Instruments, Mahwah, NJ) transmission electron microscope at 60-kV accelerating voltage.
RESULTS
In Vivo Ultrastructure
Rat. Control animals rarely had small vacuoles containing membranous figures within parenchymal and interstitial cells of heart and skeletal muscles. Rats given 300 mg
LY28l389/kg for 30 days had prominent vacuolar and membranous changes within the cytoplasm of parenchymal and
interstitial cells of the heart and skeletal muscles. Cardiac
myocytes had several membrane-bound vacuoles that con-
tained mostly concentric membranous figures or variable
amounts of electron-dense amorphous material; a few vacuoles were clear (Fig. 1). These vacuoles were in close proximity to mitochondria and myocyte nuclei. Also, many vacuoles that contained membranous figures or electron-dense
material were observed between myofilaments near T-tubules and in close proximity to mitochondria. A few clear
vacuoles and vacuoles that contained amorphous granular
accumulations were seen throughout cardiac myocytes.
Myocytes of quadriceps femoris muscle had ultrastructural changes similar to those of cardiac cells. Lysosomes
in these myocytes had variable amounts of amorphous, irregular electron-dense, or irregular or concentric lamellar membranous aggregations (Fig. 2). Some skeletal myocytes had
loss of myofilaments, were reduced in diameter, and had
cytosol largely occupied by the vacuolar and membranous
structures.
The interstitial cells, pericytes, fibrocytes, and endothelial
cells contained numerous cytoplasmic vacuolar inclusions
of membranous material similar to those observed in cardiac
and skeletal myocytes (not shown).
Dog. Control animals rarely had small vacuolated membranous figures within parenchymal and interstitial cells of
heart, skeletal muscle (quadriceps femoris), and stomach
smooth muscle. Dogs given 25 or 75 mg LY281389/kg for
LY281389-INDUCED ULTRASTRUCTURAL CHANGES
211
animals exposed to LY281389 for 14 days, affected myocytes had several variably sized lysosomes that had varying
amounts of lamellar membranous structures in close proximity to nuclei and mitochondria (Fig. 4).
In Vitro Experiments
Cellular viability measurements. Cellular viability was
greater than 80% of controls following 48-hr exposure to
LY281389 at a concentration less than or equal to 0.25 mg/
ml (Table 1). Viability was reduced to 56 and 16% of control
at 0.50 and 1.0 mg/ml, respectively.
8
FIG. 8. Transmission electron micrograph of L6 cells exposed to 0.25
mg LY281389/ml for 24 hr. Note the increased amount and size of vacuoles
that contain mostly amorphous material (arrowheads). Bar = 1 ^m.
30 days had moderate or marked vacuolar and membranous
changes, respectively, within the cytosol of parenchymal and
interstitial cells of the heart and skeletal muscles. In dogs
from the 30-day reversibility phase, changes were reduced
in severity but remained present. Within myocytes there was
a marked increase in the number of vacuoles, and the content
was highly variable. Vacuoles contained concentric lamellar
structures, had irregular membranous electron-dense material or amorphous material, were clear, or had a combination
of these conditions.
In a separate experiment, dogs exposed to 100 mg
LY281389/kg for 2, 7, or 14 days had increasing numbers
and size of membrane-bound dense concentric lamellar figures. The figures were peripheral to the nuclei of smooth
muscle cells and progressed with duration of dosing (Fig.
3). Animals exposed to LY281389 for 2 and 7 days had few
variably sized lysosomes containing membranous lamellar
figures within myocytes of stomach smooth muscle. In the
In vitro ultrastructure. L6 cells exposed to 0.25 mg
LY281389/ml had a time-dependent increase in the number
and size of vacuoles. Contents of the vacuoles were either
clear or multishaped membrane-like structures or amorphous
electron-dense material (Table 2).
L6 cells exposed to 0.25 mg LY281389 for 2.5, 5, or 10
min caused no morphological differences as compared with
untreated cell cultures; cells had occasional small clear vacuoles (Fig. 5). Cells exposed to 0.25 mg LY281389 for 20 min
had minimal vacuolar effects. There were small vacuoles
in most cells; these vacuoles were predominantly clear but
occasionally contained small membrane-like figures. Cells
exposed for 30 or 90 min or 2 hr had marked progressive
vacuolar changes that included increased number and size of
vacuoles/lysosomes and a shift in vacuole/lysosome content
from clear to membranous figures. Cells exposed to 0.25 mg
LY281389 for 30 min had a dramatic increase in number of
vacuoles/lysosomes and inclusions within vacuoles relative
to earlier times; most had a combination of amorphous granules and membrane-like figures (Fig. 6). There were few
clear vacuoles. Following 0.25 mg LY281389 exposure for
90 min, L6 cells had a moderate increase in number and
size of vacuoles, which contained mostly membrane-like
material; vacuoles rarely contained amorphous material, and
there were rare clear vacuoles. After 2 hr of LY281389
exposure, vacuolar/lysosomal contents were predominantly
small membrane-like figures. Vacuole size and number at 2
hr appeared slightly increased as compared with the 90-min
exposure.
Cells exposed to 0.25 mg LY281389 for 6 hr (Fig. 7) had
a moderate increase in the number and size of vacuoles/
lysosomes as compared with 2 hr, and after 12 hr of drug
exposure there was a dramatic increase relative to the previous time points. Vacuoles/lysosomes contained predominantly membrane-like figures, with rare clear vacuoles/lysosomes, or little amorphous or granular material.
Cell cultures exposed to 0.25 mg LY281389 for 24 or 48
hr showed prominent changes in vacuolar size and content
(Fig. 8). Cells were engorged with variably sized membranebound vacuoles containing mostly granular or amorphous
electron-dense material. These vacuoles constituted approximately 75% of the cellular volume and distorted the cell
membrane and, in many cells, the nucleus. Most cells tended
212
HORN ET AL.
FIG. 9. Transmission electron micrograph of acid phosphatase (AP)-labeled dog stomach smooth muscle exposed to 100 mg LY281389/kg for 2
days. AP label is seen confined to the penmeter of few vacuoles/lysosomes (arrowheads), but rarely is AP label found on lamellar figures (arrow). Bar
= 1 fim.
to lose their characteristic elongated appearance and assumed a "rounded" shape. After 48 hr of exposure to 0.25
mg LY281389, many cells had ruptured or perforated membranes and contained remnants of cellular organelles and
debris.
Acid Phosphatase Cytochemistry
Beagle dogs. There was no apparent AP staining in
stomach smooth muscle from control animals. Dogs that
received 100 mg LY281389/kg for 2 days had few lysosomes in stomach smooth muscle. These lysosomes were
minimally AP stained at the perimeter, but AP labeling
was rare on membranous figures in these same organelles
(Fig. 9). After 7 days, there were occasional lysosomes
in smooth muscles that had moderate AP labeling. Most
lysosomes had concentric lamellar figures, but these lamellar figures were rarely AP labeled. Dogs exposed for
14 days to 100 mg LY281389/kg had moderate numbers
of medium-sized lysosomes that were variably AP
stained; labeling ranged from none to intense, and was
located primarily at the perimeter of the lysosomes. Concentric lamellar figures, within labeled lysosomes, were
not intensely AP stained (Fig. 10).
US cell cultures. Cell cultures exposed to 0.25 mg
LY281389/ml and stained cytochemically for AP had
electron-dense label localized in lysosomes. Few small,
round lysosomes were moderately to densely stained for
AP in control cells and in cells exposed to LY281389 for
5 min to 6 hr. The incidence of densely stained lysosomes
decreased with increased duration of LY281389 exposure, and AP staining of cellular components at longer
times (12, 24, and 48 hr) was rare (Figs. 11 and 12);
however, significant numbers of enlarged lysosomes/vacuoles were present throughout exposure to the drug, as
described above.
DISCUSSION
The mechanisms by which CADs produce phospholipidosis and other lysosomotropic effects have not been
fully elaborated, but some aspects of these phenomena
have been well described (de Duve et al., 1974; LUllmann
et al, 1978; Ohkuma and Poole, 1981; Poole and Ohkuma, 1981; Reasor, 1989). CADs are known to accumulate in lysosomes (Carlier et al, 1987; Hostetler el al,
1985), although the mechanism of drug accumulation is
controversial. Proposed mechanisms include base trap-
LY281389-INDUCED ULTRASTRUCTURAL CHANGES
FIG. 10. Transmission electron micrograph of acid phosphatase (AP)labeled dog stomach smooth muscle following a 14-day exposure to 100 mg
LY281389/kg. There are several vacuoles/lysosomes containing amorphous
material and concentric lamellar figures that did not AP label (arrows). Bar
= 1 /ira.
ping of the drug in lysosomes (de Duve et al., 1974),
supported by ATP-dependent acidification of the lysosomal interior (Schneider, 1981), or, alternatively, formation of a drug complex with acidic phospholipids, leading
to accumulation of both drug and phospholipids (Liillmann et al., 1978). The structural determinants of drug
accumulation and vacuole formation have been well characterized (Poole and Ohkuma, 1981; Rorig et al., 1987).
Further, many of the biochemical consequences of lysosomal drug accumulation and vacuolization have been
reported, and include perturbations of lysosomal pH
(Klempner and Stryt, 1983; Ohkuma and Poole, 1978),
proteolysis (Wibo and Poole, 1974), and lipolysis (Hostetler et al., 1985); effects on cellular membrane fluxes
(Dean et al., 1984), such as endocytosis (Kalina and
Socher, 1991) and receptor cycling (Tietze et al., 1980;
213
Tolleschaug and Berg, 1979); and altered protein processing and transport (Hasilik and Neufeld, 1980).
The present ultrastructural study clearly demonstrated
a progressive increase in the number and size of lysosomes and in the appearance of intralysosomal material
when rats or dogs received LY281389 orally or when
cultured rat L6 skeletal muscle cells were exposed to
drug. The ultrastructural changes observed included clear
vacuoles, concentric multilamellar bodies, and vacuoles
containing amorphous material. Similar ultrastructural
changes were observed in muscle tissues from rats and
dogs receiving multiple doses of LY281389. In the dog,
the extent of ultrastructural changes increased with increasing dose and with duration of exposure. After a 30day withdrawal of drug treatment, the cellular changes
were only partially reversed; this suggests that the biological half-life of the drug in tissues was long, although a
slow reversal of ultrastructural changes could provide an
alternative explanation. The results presented here are
consistent with other observations of CAD-induced phospholipidosis, in which increased numbers of lysosomes/
vacuoles were related to increasing doses or concentrations of the CADs or to increasing duration of exposure
(Reasor, 1984; Ruben et al., 1989, 1990; Sens et al.,
1991).
The ultrastructural effects of LY281389 in rat skeletal
myoblasts were qualitatively similar to those observed in
vivo and were observed to be time dependent. Comparable
findings have been reported previously in isolated and
cultured cells (Okhuma and Poole, 1981; Ruben et al.,
1990). In addition, the effects occurred at drug concentrations that were not cytotoxic, supporting the contention
that the lysosomal effects of CADs are not necessarily
manifestations of cellular toxicity (Ruben et al., 1989).
Attempts to relate quantitatively the effects of in vitro
drug exposures to those following multiple-dose in vivo
exposures may be spurious, particularly due to pharmacokinetic factors influencing target tissue exposure; however, the lysosomotropic effects of CADs appear to be
qualitatively similar in vitro and in vivo. Because in vitro
systems respond to CADs similarly to animal models, and
have a number of experimental advantages (LUllmann et
al., 1978), cultured and isolated cell systems appear to be
a useful tool in determining the potential lysosomotropic
effects of drugs.
Acid phosphatase cytochemistry produced distinct
electron-dense labeling of lysosomes and vacuolar structures in controls and in LY281389-treated animals and
cell cultures. In dog smooth muscle, lysosomes and vacuolar structures were variably stained, but nonlamellar
membranous figures appeared to stain more intensely than
multilamellar figures. This observation may be related to
differences in the maintenance of the acidic intralysosomal environment, because lysosomal enzymes, such as
214
HORN ET AL
11
12
FIG. 11. Transmission electron micrograph of acid phosphatase (AP)-labeled L6 muscle cells exposed to 0.25 mg LY281389/ml for 5 min. Dense
AP label is seen in small lysosomes (arrows). All in vitro AP-labeled sections were not poststained with standard uranyl acetate or lead citrate, which
resulted in low-contrast micrographs. Bar = 1 fim.
FIG. 12. Transmission electron micrograph of acid phosphatase (AP)-labeled L6 muscle cells exposed to 0.25 mg LY28!389/ml for 90 min. Several
medium-sized vacuoles/lysosomes are seen without AP label, although one vacuole has AP label localized at the perimeter (arrow) Note membranous
figures and amorphous material in a few vacuoles (arrowheads). Bar = I /jm
LY281389-INDUCED ULTRASTRUCTURAL CHANGES
AP, have acidic pH optima (Mellman et a/., 1986). It has
been shown that inhibiting lysosomal phospholipases A
and C contributes to accumulation of phospholipid-drug
complex and leads to the appearance of multilamellar
structures (Hostetler et al., 1985). AP staining at the periphery of lysosomes was occasionally observed following drug exposure and a similar finding has previously
been reported (Koizumi et al., 1986). Similar cytochemical staining was seen in myoblast cells, although after
prolonged exposure a progressive loss of AP staining was
observed, suggesting an inability of the cell to maintain
an acidic lysosomal pH and, therefore, AP activity. Alternatively, the amounts of AP protein present in lysosomes
may have declined with prolonged drug exposure.
In summary, LY281389 caused formation of cytoplasmic vacuoles in cultured L6 myoblast cells and in
cardiac, smooth, and skeletal muscles of rats and dogs
receiving multiple doses of drug. Myoblasts showed timedependent increases in the sizes and numbers of vacuoles,
particularly those containing membranous material. Rat
and dog muscle showed similar ultrastructural changes,
with frequent appearance of concentric multilamellar
bodies. These effects in vivo were partially reversed when
compound was withdrawn for 30 days. AP cytochemistry
indicated the involvement of lysosomes in formation of
cytoplasmic vacuoles in vitro and in vivo.
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