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[CANCER RESEARCH 43, 646-652, February 1983] 0008-5472/83 / 0043-0000502.00 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 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. 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. FEBRUARY 1983 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. 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. REFERENCES 1. Cheng, H., and Leblond, C. P. Origin, differentiation, and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J. Anat., 141: 537-562, 1975. 2. Corti, A., Dave, C., Williams-Ashman, H. G., Mihich, E., and Schenone, A. Specific inhibition of the enzyme decarboxylation of S-adenosylamethionine 5 A. Pleshkewych and C. W. Porter, unpublished observations. 648 by methylglyoxal-bis(guanylhydrazone) and related substances. Biochem. J., 139: 351-357, 1974. 3. Dave, C., and Caballes, L. Studies on the uptake of methylglyoxal-bis(guanylhydrazone) (CH~-G) and spermidine in mouse leukemia sensitive and resistant to CH3-G. Fed. Proc., 32: 736, 1973. 4. Freireich, E. J., Frei, E., III, and Karon, M. Methylglyoxal-bis(guanylhydrazone) (methyl GAG), a new active agent against adult leukemia. Cancer Chemother. Rep., 16:183-186, 1962. 5. Knight, W. A., III, Livingston, R. B., Fabian, C., and Costanzi, J. Phase I-II trials of methyl-GAG: southwest oncology group pilot study. Cancer Treat. Rep., 63: 1933-1937, 1979. 6. Kramer, D. L., Wiseman, A., and Porter, C. W. Characterization of VA~-cell vairants resistant to the antiproliferative effects of methylglyoxalbis(guanylhydrazone). Proc. Am. Assoc. Cancer Res., 22: 30, 1981. 7. Levin, R. H., Henderson, E., Karon, M., and Freireich, E. J. Treatment of acute leukemia with methylglyoxal-bis-guanylhydrazone(methyl GAG). Clin. Pharmacol. Ther., 6 : 3 1 - 4 2 , 1964. 8. Mihich, E. Prevention of the antitumor activity of methylglyoxal-bis-(guanylhydrazone) (CH3-G) by spermidine. Pharmacologist, 5: 270, 1963. 9. Mihich, E. The bis(guanylhydrazones). In: A. C. Sartorelli and D. G. Johns (eds.), Handbook of Experimental Pharmacology, New Series, Vol. 38/2, pp. 766-788. New York: Springer Verlag, 1975. 10. Mikles-Robertson, F., Feurstein, B. F., Dave, C., and Porter, C. W. The generality of methylglyoxal-bis-(guanylhydrazone)-induced mitochondrial damage and the dependence of this effect on cell proliferation. Cancer Res., 39: 1919-1926, 1979. 11. Pathak, S. N., Porter, C. W., and Dave, C. Morphological evidence for an antimitochondrial action by methylglyoxal-bis(guanylhydrazone). Cancer Res., 37: 2246-2250, 1977. 12. Pleshkewych, A., Kramer, D. L., Kelly, E., and Porter, C. W. Independence of drug action on mitochondria and polyamines in L1210 leukemia cells treated with methylglyoxal-bis(guanylhydrazone). Cancer Res., 40: 45334540, 1980. 13. Porter, C. W., Dave, C., and Mihich, E. Inhibition of S-adenosylmethionine decarboxylase as an approach in cancer therapeutics. In: D. Morris and L. Marton (eds.), Perspectives and Issues in Polyamine Research, pp. 407436. New York: Marcel Dekker, Inc., 1981. 14. Porter, C. W., Dworaczyk, D., Ganis, B., and Weiser, M. M. Polyamines and biosynthetic enzymes in the intestinal mucosa of the rat and the influence of methylglyoxal-bis(guanylhydrazone).Cancer Res., 40: 2330-2335, 1980. 15. Porter, C. W., Mikles-Robertson, F., Kramer, D., and Dave, C. Correlation of ultrastructural and functional damage to mitochondria of ascites L1210 cells treated in vivo with methylglyoxal-bis(guanylhydrazone)orethidium bromide. Cancer Res., 39: 2414-2421, 1979. 16. Regelson, W., and Holland, J. F. Clinical experiences with methylglyoxalbis-(guanylhydrazone) HCI, a new agent with clinical activity in acute myeIocytic leukemia and the lymphomas. Cancer Chemother. Rep., 2 7:15-26, 1963. 17. Siimes, M., Seppanen, P., Alhonen-Hongisto, L., and Janne, S. Synergistic action of two polyamine antimetabolites leads to rapid therapeutic response in childhood leukemia. Int. J. Cancer, 28: 567-570, 1981. 18. Warrell, R. P., Lee, B. J., Kempin, S. J., Lacher, M. J., Strauss, D. J., and Young, C. W. Effectiveness of methyl-GAG [methylglyoxal-bis(guanylhydrazone)] in patients with advanced malignant lymphomas. Blood, 5 7: 1011-1014, 1981. 19. Williams-Ashman, H. G., and Schenone, A. Methylglyoxal-bis(guanylhydrazone) as a potent inhibitor of mammalian and yeast S-adenosyl methionine decarboxylases. Biochem. Biophys. Res. Commun., 46: 288-295, 1972. CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. Changes in Mitochondria Treated with MGBG %~i~i~!i i i I!!!))Ui;L, dii:~]i!~84 ~il;;;'84184 ...... .... iiii, !==:!i!!i! =;==i~ ~,~i~,i~~i ,iiiill~ii~!~!ii!~i! 'd ii@i .i i iiii=o iiiiiiii ~i~I~i!~i84i~!i;i?~84184 )!~!!i i84 ~;~i84 lili i~84 i i!i ~'~~ii~i~i~ii~i~!il~i i~i~i!~;~i~i'i~i~i!'~il~'ii~ilili: ....!!!!/~;~ ...... ~ii i!;/i! m 84 84 ,iii,i84 ii!ii!84:!'!!'~ ',~#,i~i!~!~i~',~ii~iii~ i~!i~iiiii~i~i~iii~~?~ ~i~iiii~i~i~ii~!'!!!!! ))~ m i i ! iiijjii]i iiii i r i! its ~ ., !ii~/iiiiiiiT" "! ......... i = =i=ii=i! .......................... iiii i!= =i=i:=ii=ii E;==]II",==I:I === ,; ,,==,~ ) i&'i]!i; I ~~T! = ~iiii ;ik i11 !! iiiiiiiiJJiJii~ l!ir I~-'+'!IIf!I;=,~<,=;EII!. ! ==,. . . . . . . . ~ 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 . FEBRUARY1983 649 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. A. Pleshkewych et al. ~..... ~ii!i~:i~i~iiii~,i,~i,i,,,i~iili i ilia,:!~!!i84 !!~!'~i~i~!!~!~,~i~i,~........ ,~ i~~,,~~ ~,~i '!!!~!'!?!!!!!~i!i~!~!~' ! !i~i~i~~iii~ii~i,i~ii~i~,~ii~iii~i,ili~iii~iii~iiiiiii~ii!!ii~!ii~iiii~i!i:!~! il, 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 CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. 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). FEBRUARY1983 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. 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 CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. 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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