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[CANCER RESEARCH 43, 646-652, February 1983]
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Ultrastructural Changes in the Mitochondria of Intestinal Epithelium of
R o d e n t s T r e a t e d with M e t h y l g l y o x a l - b i s ( g u a n y l h y d r a z o n e ) 1
A. P l e s h k e w y c h , T. C. Maurer, and Carl
W.
Porter 2
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New
York 14263 [T. C. M., C. W. P.], and Department of Natural Sciences, Daemen College, Amherst, New York 14226 [A. P.]
ABSTRACT
Ultrastructural studies of rats or mice treated for 24 hr with
a toxic dose ( 1 0 0 m g / k g ) of m e t h y l g l y o x a l - b i s ( g u a n y l h y drazone) revealed the presence of d a m a g e d m i t o c h o n d r i a in
the crypt cells of the intestinal epithelium. M i t o c h o n d r i a w e r e
severely swollen and electron lucent, and a p p e a r e d to be
similar to those observed previously in a variety of cell types
treated in v i t r o and in v i v o with m e t h y l g l y o x a l - b i s ( g u a n y l h y drazone). Since thymidine incorporation into the intestine
was not found to be decreased until after 24 hr, it is c o n c l u d e d
that the mitochondrial d a m a g e of m e t h y l g l y o x a l - b i s ( g u a n y l hydrazone) could be responsible for the antiproliferative toxicities of the drug.
INTRODUCTION
Early clinical trials with M G B G 3 revealed impressive antitumor activity that was unfortunately offset by prohibitive toxic
reactions, including fetal hypoglycemia, m y e l o s u p p r e s s i o n ,
and severe ulcerations of the skin and gastrointestinal tract (4,
7, 18). A c c e p t a b l e reversible toxicity was eventually achieved
by administering the drug on weekly, rather than daily, infusion
schedules (5). Since then, M G B G has u n d e r g o n e a " s e c o n d
l o o k " as a clinical anticancer agent, and good activity has been
reported in the treatment of certain neoplasias with the drug as
a single agent (18) or in combination with the inhibitor of
polyamine biosynthesis, ~x-difluoromethylornithine (1 7).
Of the 2 intracellular sites of M G B G action, interference with
mitochondrial function (1 1, 12) and depletion of certain polyamines by inhibition of S - a d e n o s y l m e t h i o n i n e d e c a r b o x y l a s e
(2, 19), it a p p e a r s from our studies that the f o r m e r is most
critically involved in at least the early antitumor effects exerted
by the drug (1 1, 12). 4 The limited selectivity of M G B G for
proliferating neoplastic tissues may be afforded by its shared
uptake into cells with polyamines, s p e r m i d i n e in particular (3,
13).
Because of the r e n e w e d clinical interest in M G B G , it is
important to determine the basis for the gastrointestinal toxicity
which still appears on the w e e k l y infusion schedule. Previously,
we have d e t e r m i n e d that polyamine pools are not substantially
diminished in the intestinal epithelium of mice treated with
1 This investigation was supported by Research Grant CA 22153 and Core
Grant CA 24538 from the National Cancer Institute, Department of Health,
Education and Welfare.
2 To whom requests for reprints should be addressed.
3 The abbreviation used is: MGBG, 1,1 '-[(methylethanediylidene)-dinitrilo]-diguanine, also known as methylglyoxal-bis(guanylhydrazone), MethyI-G or MethylGAG.
4 A. Wiseman, D. Kramer, and C. W. Porter. Human VA2 cell variants resistant
to the antiproliferative effects of methylglyoxal-bis(guanylhydrazone), submitted
for publication.
Received February 18, 1982; accepted October 21, 1982.
646
M G B G by the time that intestinal toxicity appears (14). In the
present study, we have examined the ultrastructure of the
gastrointestinal epithelium of rats and mice treated with M G B G
to determine w h e t h e r the mitochondria are affected as they are
in t u m o r cells treated in v i t r o (10, 1 1 ) or in v i v o (5, 12) with the
drug. The results indicate that M G B G alters p r o f o u n d l y the
ultrastructure of those mitochondria located in the crypt cells
of the intestinal epithelium prior to inhibition of [3H]thymidine
incorporation.
MATERIALS AND METHODS
DBA/2J mice, weighing 20 to 25 g, were given a single i.p. injection
of MGBG (100 mg/kg) in sterile 0.85% NaCI solution. This dose is
slightly below the 50% lethal dose for MGBG in rodents (100 to 120
mg/kg) and is known to cause diarrhea (9). Animals receiving sterile
0.85% NaCI solution alone served as controls. After 24 hr, the mice
were sacrificed by cervical dislocation, and the abdominal cavity was
opened. One end of the jejunum was transected, and after a section of
intestine was clamped, the lumen was perfused with oxygenated phosphate-buffered (23 ml 0.2 M Na2PO4-H20, 77 ml 0.2 M Na2HPO4, 100
ml distilled water, and 0.5 ml 1% CaCI2) 3% glutaraldehyde (pH 7.4,
480 mOsmol) at 4 ~ Portions of the jejunum were then excised, placed
in glutaraldehyde, and cut into small blocks.
Similarly, Sprague-Dawley rats weighing 200 to 250 g were given a
single i.p. injection of MGBG (100 mg/kg) suspended in sterile 0.85%
NaCI solution. Control rats were given injections of sterile 0.85% NaCI
solution. After 24 hr, the fasted rats were anesthetized with ether, and
the tissue samples were removed as described for mice.
Tissue blocks from the intestines of mice and rats were fixed for 24
hr at 4 ~ in phosphate-buffered 3% glutaraldehyde. The fixed tissue
blocks were then washed in phosphate buffer overnight, postfixed in
phosphate-buffered 1% osmium tetroxide, dehydrated in a graded
alcohol series, and embedded in Epon-Araldite resin mixture. Semithin
sections ( - 5 0 0 nm) were prepared for light microscopy with a PorterBlum MT-1 ultramicrotome (Sorvall Corp., Norwalk, Conn.) and stained
with 1% aqueous toluidine blue containing 1% sodium borate. Blocks
were selected and trimmed to show longitudinal sections through the
villus, including the crypt regions. Thin sections (~90 nm) were stained
sequentially with uranyl acetate and lead citrate and then examined
with a Siemens Elmiskop 101 electron microscope at 80 kV.
As an indication of the antiproliferative effects of MGBG on the
intestine, the incorporation of [3H]thymidine into various regions of the
intestine was measured in mice bearing L1210 cells. Mice were first
inoculated i.p. with 106 L1210 cells 3 days prior to drug treatment.
They were then given a single i.p. injection of MGBG (100 mg/kg) and
0 (control); 24 or 48 hr later, they were given i.p. injections of 500/~Ci
of [3H]thymidine (40 Ci/mmol; Amersham Corp., Arlington Heights, III.)
in sterile 0.85% NaCI solution. After 1 hr, the L1210 cells and segments
of the intestine were removed, washed in 0.85% NaCI solution, and
homogenized on ice for 2 to 4 min with a Polytron (Brinkman Instruments, Westbury, N. Y.). The homogenate was precipitated with 1%
phosphotungstJc acid in 0.5 N HCI and washed twice in 10% phosphotungstic acid by centrifugation. The final precipitate was completely
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Changes
dissolved at 45 ~ in Soluene 350 (Packard Instrument Co., Inc., Downers Grove, II1.) and placed in scintillation counting fluid. Vials were
counted in a Packard Model 3320 scintillation counter. Data were
expressed as dpm per mg protein per hr.
RESULTS
Treatment of mice with 100 m g / k g for 24 hr induced definite
signs of drug toxicity as indicated by relative inactivity, diarrhea, piloerection, and death in - 1 0 % of the animals. Of the
1 2 MGBG-treated mice which were examined morphologically,
9 mice showed drug-induced changes in their intestinal epithelium. Examination of semithin sections of jejunum at the light
microscope level revealed the presence of numerous patent
vacuoles in the cytoplasm of cells located in LieberkLihn's
crypts (Fig. 1 ). The vacuoles varied in size and appeared to be
different from the secretory granules or mucin vacuoles, which
were densely stained. The vacuoles were seen in the crypt
cells of the jejunum but not in the cells of the villus walls (Fig.
1 ) or in the crypt cells of untreated mice (Fig. 2, A and B).
At the electron microscope level, the vacuoles of the crypts
were observed to correlate with distended mitochondria. Its
appearance (Fig. 2, C and D) was nearly identical to that
described previously by this laboratory for a variety of cell
types treated in vitro 4 (10, 1 1 ) or in vivo (1 2, 1 5) with MGBG.
The mitochondria were swollen, their cristae were disoriented,
and the matrix appeared to be diffuse and electron lucent.
Other cellular organelles and the nuclear substructure were
not affected by MGBG treatment. All of the cells of the crypts
seemed to be equally affected, including those undergoing
mitosis and those containing mucin or secretory granules (Fig.
2D). When the intestines were examined 36 or 48 hr after the
injection of MGBG, swollen mitochondria were not apparent in
the crypt cells or in the villous cells, suggesting that the effect
had reversed itself morphologically. However, many of the
mitochondria of both crypt and villous cells contained electrondense granules in their matrix.
In rats treated with MGBG, similar morphological effects
were observed in the intestinal epithelium. The drug effect,
however, was observed much more consistently, being present
in all of 4 rats examined. As with the mouse, the cytoplasmic
vacuolation was confined to the cells of the crypts and was not
seen in the cells of the villus wall or in the crypt cells of
untreated animals (Fig. 3, A and B). The mitochondria of the
crypt cells, by electron microscopy, appeared swollen, as
described for mice (Fig. 3, C and D), but differed in that many
of the organelles contained granular electron-dense inclusions
(Fig. 4). Similar inclusions have been described in cultured
human cells treated with MGBG (6, 10). 4
The ultrastructure of the liver, kidney, and spleen from
MGBG-treated rodents was also examined, but neither mitochondria nor other structures appeared to be affected.
The [3H]thymidine incorporation data are summarized in Table 1. In keeping with the rapid growth characteristics of ascites
tumors, the L 1 2 1 0 cells were found to incorporate approximately 100 to 1000 times more thymidine than did any region
of the intestine on a d p m / m g / h r basis. The small intestine was
about 2-fold more active than the colon in this regard. Incorporation into L 1 2 1 0 cells decreased by 45% at 24 hr after
MGBG injection and then returned to control levels by 48 hr.
By contrast, thymidine incorporation in the intestine was slower
in M i t o c h o n d r i a
Treated
with
MGBG
Table 1
Incorporation of [3H]thymidine into intestine and L 1210 leukemia cells following
MGBG treatment
dpm/mg protein/hr x 10 3 at time following MGBG
injection
Tissuea
0 hr
24 hr
48 hr
L1210 cells
3676
2068
4046
Dundenum
25.8
27.8
16.7
Jejunum
31.4
31.6
15.2
Ileum
25.2
24.2
13.4
Colon
10.9
12.2
10.0
a On the basis of 2 experiments with 3 mice at each time point.
to occur and was not apparent until 48 hr after MGBG injection.
Incorporation into the small intestine was more inhibited by the
drug, while that into the colon was virtually unaffected.
DISCUSSION
Selective ultrastructural damage was apparent in the crypt
cells of the intestinal epithelium 24 hr after a single injection of
MGBG (100 m g / k g ) . Since [3H]thymidine incorporation into
the small intestine was not apparent until after 24 hr, the
mitochondrial effect of MGBG could be responsible for its
antiproliferative action. Although [3H]thymidine incorporation
into L1 210 cells was already inhibited by 24 hr, we have shown
previously (1 2) that mitochondrial damage is apparent in 85%
of these cells by 12 to 16 hr. The above findings are in keeping
with what has been found in in vitro systems (10, 1 1 ). Namely,
mitochondrial damage precedes detectable inhibition of cell
growth, and its time of onset seems to be related directly to the
proliferative activity of the cells or tissues.
Subacute toxicities by MGBG have been divided into proliferative (gastrointestinal, bone marrow, and lymphoid) and nonproliferative (hepatic, renal, and cardiac) types (9). On the
basis of the present findings linking mitochondrial damage to
gastrointestinal toxicity and previous studies showing mitochondrial damage in subconfluent cells or lymphocytes undergoing blastogenesis (10), it seems that in general the proliferative toxicities related to MGBG are the result of drug
interference with mitochondrial function. Prevention of the proliferative toxicities of MGBG by coadministration of spermidine
(8) is probably due to preferred uptake of spermidine over
MGBG by a shared carrier mechanism (3, 13) as opposed to
prevention of drug depletion of spermidine pools by MGBG
inhibition of S-adenosyI-L-methionine decarboxylase (2, 19).
The finding that the MGBG-induced mitochondrial damage
to intestinal epithelium is confined to the cells of the crypts is
consistent with a previous study showing that only proliferating
cells are affected by the drug (10) and could be the result of
increased drug uptake by dividing cells. The cells located in
the base of the crypts are mitotically active and poorly differentiated. According to the report of Cheng and Leblond (1),
these multipotential cells give rise to villous, columnar, mucous,
enteroendocrine, and Paneth cells which, excepting the Paneth
cells, experience a differentiation process as they move towards the villus tip. This could be the reason that all cells in the
crypts, including those which appeared to be differentiated and
nondividing, contained damaged mitochondria. Apparently, the
damaged mitochondria rapidly recover normal ultrastructure as
circulating MGBG levels decrease, since they were not apparent in any of the epithelial cells by 36 hr after drug injection.
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647
A.
Pleshkewych
et
al.
T h e u l t r a s t r u c t u r a l c h a n g e s i n d u c e d in t h e m i t o c h o n d r i a o f
the intestinal epithelium of mice and rats treated with MGBG
a r e v i r t u a l l y i d e n t i c a l to t h o s e o b s e r v e d in L1 21 0 c e l l s t r e a t e d
in v i v o w i t h t h e d r u g ( 1 2 ,
15). In t h e l a t t e r , w e h a v e d e m o n strated that interference with mitochondrial function precedes
b y 2 h r s i g n i f i c a n t r e d u c t i o n in s p e r m i d i n e o r s p e r m i n e p o o l s
a s a c o n s e q u e n c e of M G B G i n h i b i t i o n o f S - a d e n o s y l - L - m e t h i onine decarboxylase (12). Mitochondrial effects by MGBG,
t h e r e f o r e , a r e p r o b a b l y d u e to a d i r e c t i n t e r a c t i o n w i t h t h e
o r g a n e l l e a n d a r e n o t r e l a t e d to t h e i n h i b i t i o n o f p o l y a m i n e
biosynthesis. Moreover, the aromatic bis(guanylhydrazone),
4,4'-diacetyldiphenyl urea bis(guanylhydrazone) (also known
as DDUG), which has no effect on polyamines, does not prod u c e m i t o c h o n d r i a l d a m a g e to t h e c e l l s o f t h e i n t e s t i n a l e p i t h e lium s e v e n t h o u g h , l i k e M G B G , it c a u s e s e x t e n s i v e m i t o c h o n d r i a l d a m a g e in L1 21 0 c e l l s .
It n o w a p p e a r s f r o m r e c e n t s t u d i e s w i t h L1 21 0 c e l l s (1 2) a n d
w i t h M G B G - r e s i s t a n t v a r i a n t s o f VA2 c e l l s (1 6), 4 t h a t m i t o c h o n drial effects by MGBG are probably responsible for the antiproliferative action of the drug. When taken together with earlier
studies (14), the present studies suggest that mitochondrial
effects by MGBG are also responsible for the gastrointestinal
t o x i c i t y of t h e d r u g . P r e v i o u s l y , w e f o u n d t h a t f o l l o w i n g M G B G
t r e a t m e n t , t h e p o l y a m i n e p o o l s of t h e i n t e s t i n a l e p i t h e l i u m a r e
not significantly depleted by the time that intestinal toxicity
appe~r.~ ( 1 4 ) . A s s h o w n h e r e , t h e m i t o c h o n d r i a a r e c l e a r l y
d a m a g e d at t h i s t i m e , s u g g e s t i n g t h a t i n t e r f e r e n c e w i t h m i t o chondrial function rather than polyamine biosynthesis gives
rise to at l e a s t o n e o f t h e p r o l i f e r a t i v e t o x i c i t i e s of M G B G .
ACKNOWLEDGMENTS
The authors gratefully acknowledge the skilled technical assistance of Deborah Ogden and John Miller.
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Changes in Mitochondria Treated with MGBG
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Fig. I. Light micrograpb of a semithJn s e c t i o n s h o w i n g a villus b a s e of intestinal epithelium taken from the jejunum of a m o u s e treated for 1 4 br with MGBG (I 0 0
mg/kg). Note the presence of numerous patent vacuoles located in the cytoplasm of crypt cells (C). The cells of the villus (V) are not vacuolated. • 2 0 4 7 .
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A. Pleshkewych et al.
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Fig. 2. Light and electron micrographs of intestinal crypt cells taken from the jejunum of untreated mice (A, C) and mice treated for 24 hr with M G B G (100 m g / k g )
(B, D). The crypts of untreated mice (A) lack the vacuoles seen in MGBG-treated mice (B). A, x 800; B, x 800; C, x 6300; D, 5300.
650
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VOL.
43
Changes in Mitochondria Treated with MGBG
Fig. 3. Light and electron micrographs taken from the intestinal crypts of untreated (A, C) and MGBG-treated rats (B, D). As observed in mice (Fig. 2), the
cytoplasmic vacuoles in the crypts (B) correlate with distended mitochondria (D). All cells and all mitochondria appear to be uniformly affected. A, x 800; B, x 800;
C, • 6300; D, x 5300).
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651
A. Pteshkewych et al.
Fig. 4. Granular intramitochondrial inclusions observed in the intestinal crypt cells of rats treated for 24 hr with MGBG (100 m g / k g ) x 20,000.
652
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VOL.
43
Ultrastructural Changes in the Mitochondria of Intestinal
Epithelium of Rodents Treated with
Methylglyoxal-bis(guanylhydrazone)
A. Pleshkewych, T. C. Maurer and Carl W. Porter
Cancer Res 1983;43:646-652.
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