Toxicology, 74 (1992) 223-232 Elsevier Scientific Publishers Ireland Ltd.
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Benznidazole-induced ultrastructural alterations in rat adrenal cortex. Mechanistic studies C.R. de Castro, E.G. Diaz de Toranzo and J.A. Castro Centro de Investigaeiones Toxieol6gieas (CE1TOX) CITEFA/CON1CET Zufriategui 4380. 1603 Villa Martelli, Prov. de Buenos Aires (Argentina)
(Received February 1lth, 1992; accepted June 10th, 1992)
Summary Benznidazole (Bz) (N-benzyl-2-nitro-l-imidazole acetamide) is a drug used against Chagas' disease, a parasitic disease afflicting several millions of Latin Americans. Bz administration to Sprague-Dawley male rats at 100 mg/kg p.o. caused subcellular alterations in the adrenal cortex involving fasciculata and reticularis zones but not in the glomerulosa. There is Bz nitroreductase activity in the adrenal microsomal and mitochondrial fractions but most of it is localized in mitochondria. Activity in the two fractions requires NADPH under anaerobic conditions. Mitochondrial Bz nitroreductase activity was inhibited by oxygen. A minor but statistically significant inhibition was observed in mixtures incubated under carbon monoxide. Microsomal Bz nitroreductase activity was not detected under oxygen atmosphere and was not inhibited under carbon monoxide. No Bz nitroreductase activity mediated by xanthine oxidase or aldehyde oxidase was detected in the cytosolic fraction from rat adrenals. Electron microscopic examination of the adrenal cortex from Bz-treated animals revealed cells with marked lipid accumulation and alterations in nuclei, endoplasmic reticulum and mitochondria in the reticularis and fasciculata zones. In vitro results suggest a Bz nitroreductive activation, with minor or null P-450 participation, leading to reactive metabolites able to cause damage in various organelles. Key words: Benznidazole; Fasciculata and Reticularis Zones; Adrenals; Chagas' disease
Introduction B e n z n i d a z o l e (Bz) is a d r u g p r e s e n t l y in use in the c h e m o t h e r a p y o f C h a g a s ' disease, a p a r a s i t i c disease a f f l i c t i n g several m i l l i o n p e o p l e in L a t i n A m e r i c a [ 1 - 3 ] . Bz t h e r a p y , h o w e v e r , has s i g n i f i c a n t u n d e s i r a b l e side effects w h i c h f r e q u e n t l y cause p a t i e n t s to s t o p t r e a t m e n t [2,3]. B o t h c h e m o t h e r a p e u t i c a n d toxic side effects o f Bz a p p e a r to be r e l a t e d to Bz r e d u c t i v e b i o t r a n s f o r m a t i o n [4-12]. In p r e v i o u s studies w e r e p o r t e d the o c c u r r e n c e o f u l t r a s t r u c t u r a l a l t e r a t i o n s in diff e r e n t rat tissues r e l a t e d to Bz n i t r o r e d u c t a s e a c t i v i t y [11,12]. Bz n i t r o r e d u c t a s e activity was p r e v i o u s l y r e p o r t e d to be a s s o c i a t e d w i t h the p r e s e n c e o f c y t o c h r o m e P-450 (P-450), P - 4 5 0 r e d u c t a s e , x a n t h i n e o x i d a s e a n d a l d e h y d e o x i d a s e [2,3,5,11,12]. Correspondence to: C.R. de Castro, Centro de Investigaciones Toxicol6gicas (CEITOX) CITEFA/CONICET. Zufriategui 4380, 1603 Villa Martelli, Prov. de Buenos Aires, Argentina.
0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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This work describes ultrastructural alterations in adrenal cortex from rats pretreated with Bz and their relation to the Bz nitroreductase activity in several cellular fractions. Materials and methods
Chemicals Bz (N-benzyl-2-nitro-l-imidazole acetamide) was a gift from Hoffman La Roche & Co. Ltd. All other chemicals used were of analytical grade. Animals and treatments Sprague-Dawley male rats (220-250 g) bred in our laboratory were used throughout these experiments. The animals were housed in wire cages. Temperature in the animal room was 23 + 2°C and the relative humidity was between 35 and 65%. Light was on from 06:00 h-18:00 h. Animals had access to food (Purina Chow rat diet) and water at all times during the experiments. Bz was given orally as a single dose of 100 mg/kg suspended in 1% carboxy methylcellulose (CMC). The control group received vehicle only. Adrenal glands from anesthetized animals dosed with 50 mg/kg body wt. pentobarbital were removed 24 h after Bz administration. The rats were then sacrificed without delay. Adrenals isolated from untreated animals bred under the same conditions were used for the in vitro studies. The rats were sacrificed and the adrenal glands rapidly excised and processed. Isolation of subcellular fractions Adrenal glands were homogenized in a Teflon-glass Potter-Elvehjem homogenizer with 10 vols. of 1.15% KC1. The homogenate was centrifuged for 10 min at 600 × g and after the pellet was resuspended in 10 vols. of 1.15% KC1 a further centrifugation under the same conditions followed. The resultant supernatants were collected and centrifuged for 15 min at 8000 × g. The pellet obtained was the mitochondrial preparation which was washed once with 20 vols. of 1.15% KC1 before use. The remaining supernatants were centrifuged again for 1 h at 105 000 × g in order to obtain the pelleted microsomes and the supernatant cytosol fraction. This fraction was dialyzed overnight in the cold room against 2 1 of 20 mM phosphate buffer (pH 7.4). All these operations were performed at 2-4°C. Enzymatic and chemical determinations All incubations were done in 20 ml septum-stoppered vials at 37 °C. In a final volume of 2.5 ml of 20 mM phosphate buffer (pH 7.4) vials contained: (a) the subcellular fraction (either a final concentration ranging from 0.49 to 1.40 mg protein/ml of mitochondria, or 0.25 to 0.64 mg protein/ml of microsomes, or 0.96 to 1.11 mg protein/ml of cytosol); (b) 0.05 ml NADPH-generating system composed by: 0.3 M Tris-HC1 buffer (pH 7.4), 0.2 ml; 1 M MgCI2, 0.2 ml; isocitric acid dehydrogenase type IV from porcine heart, 0.6 ml; dl-isocitric acid trisodium salt, 124 mg; N A D P H sodium salt, 20 rag; and (c) Bz to a final concentration 0.288 raM. For cytosol incubations N A D P H was omitted and either N-methylnicotinamide (2.5 mM) or hypoxanthine (0.25 mM) as substrate was included. Vials containing the
225
different subcellular fraction suspensions were bubbled with oxygen-free nitrogen, pure oxygen or carbon monoxide for 5 min prior to the addition of N A D P H and Bz as a dimethylformamide solution. The final concentration of dimethylformamide in the incubation mixture was 2%. After incubation for 30 or 60 min reactions were terminated by the addition of 1 ml of 15% zinc sulfate. Incubation mixtures were added to 2.5 g of NaC1 and extracted with 7.0 ml of ethylacetate. The organic phase was read in a spectrophotometer at 315 nm. At this wave length the drug has maximum absorption. Absorbance was compared against a calibration curve of different concentrations of Bz in ethyl acetate. Enzymatic activity was determined by measuring the decrease in absorbance due to the reduction of the Bz nitrogroup. Absorbance values at 30 or 60 min of incubation were subtracted from the absorbance value at zero incubation time taken as a blank. This procedure compensates for the absorbance of endogenous substances other than the substrate. Under these experimental conditions Bz nitroreductase activity was linear with time and protein content.
Electron microscopy Both adrenals from each rat (5 animals per group) were prepared for electron microscopy. Ten cubes per adrenal gland of approximately 1 mm were cut under 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), fixed by immersion for 24 h, and fixed in 1% osmium tetroxide. The cubes were immersion-stained in block with uranyl acetate, dehydrated in graded ethanol solutions and propylene oxide and later embedded in epoxy resine [16]. Thick sections for orientation purpose were obtained with a LKB ultratome using glass knives and stained with toluidine blue. Thin sections were cut with diamond knives, stained with uranyl acetate [17] and lead citrate [18] and examined in a Philips EM 300 electron microscope. Statistics A decision tree for selecting appropriate statistical procedures (parametric or nonparametric) was applied to results from every experiment [ 111. Significance between two groups was established in all cases using F-test followed by Student's t-test.
TABLE 1 BENZNIDAZOLE N I T R O R E D U C T A S E ACTIVITY IN D I F F E R E N T CELLULAR FRACTIONS FROM RAT ADRENALS a Values are the means ± S.D. of three different experiments. Each result was obtained by pooling the adrenal glands from the number of rats indicated in parentheses. Subcellular fraction
Bz nitroreductase activity nmol Bz/min per mg protein
Mitochondria Microsomes
2.775 ~- 0.200 (22) 1.338 ± 0.091 (22)
aThe different subcellular fractions were incubated at 37°C under a N 2 atmosphere with Bz and NADPH generating system for 30 min in case of mitochondria and for 60 min in case of microsomes.
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Results
Bz nitroreductase activity in different cellular fractions from rat adrenals Mitochondria and microsomes from adrenal glands exhibited Bz nitroreductase activity under anaerobic conditions and in the presence o f N A D P H (Table I). Most o f the activity was f o u n d in the mitochondrial fraction. W h e n the cytosolic fractions were incubated at 37°C for 60 min under a nitrogen atmosphere with Bz and either N-methyl-nicotinamide or hypoxanthine as substrate, xanthine oxydase (0.038 4- 0.03 nmol Bz/min per mg P) and aldehyde oxydase (0.026 ± 0.045 nmol Bz/min per mg P) activities were negligible.
Mitochondrial and microsomal Bz nitroreductase activity from rat adrenals under different atmospheres Mitochondrial Bz nitroreductase activity was almost completely abolished when incubated under oxygen atmosphere. A statistically significant inhibition was observed when incubated with pure c a r b o n monoxide. Microsomal Bz nitroreductase activity was not detected when the fraction was incubated under pure oxygen and no differences were observed between carbon monoxide and nitrogen incubations (Table II).
Bz-induced ultrastructural changes in adrenal cortex The glomerulosa, fasciculata and reticularis zones were examined and since Bzinduced damage was confined exclusively to the zones fasciculata and reticularis, description o f alterations will be confined to these two zones. The fasciculata cells from control rats were rich in smooth endoplasmic reticulum membranes and mitochondria. The smooth endoplasmic reticulum was vesicular and arranged in layers surrounding lipid droplets and mitochondria. Mitochondria were numerous,
TABLE II BENZNIDAZOLE NITROREDUCTASE ACTIVITY IN DIFFERENT CELLULAR FRACTIONS FROM RAT ADRENALS UNDER DIFFERENT ATMOSPHERES a Values are the means 4- S.D. of three different experiments. Each results was obtained by pooling the adrenal glands from the number of rats indicated in parentheses. Subcellular fraction
Atmosphere
Bz nitroreductase activity nmoles/min/mg protein
Mitochondria
Nz 02 CO
2.830 4- 0.033 (69) 0.026 4- 0.032b (69) 2.266 4- 0.191b (69)
Microsomes
N2 02 CO
1.126 + 0.590 (53) 0 b (53) 1.103 4- 0.419 (53)
alncubation mixtures were as in Table I except for the different atmospheres. bp < 0.05 compared with the N 2 atmosphere.
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Fig. 1. An electron micrograph ( × 8800) from the fasciculata zone of a control rat adrenal showing round nucleus, abundant spherical mitochondria, numerous free ribosomes and polysomes and well developed Golgi.
Fig. 2. Electron micrograph ( x 14 000) from the fasciculata zone of a control rat adrenal. In some cells, the cytoplasm exhibits numerous normal-looking mitochondria and a deep mvagination occupied by a lipid droplet.
228
Fig. 3. Reticularis zone of a control rat adrenal showing a round nucleus, numerous mitochondria with lamellar and tubular cristae and the presence of lipofuscin granules in the cytoplasm ( x 8800),
Fig. 4. Electron micrograph ( x 14 000) from the fasciculata zone of a benznidazole-treated rat adrenal. The nuclear membrane and the Golgi exhibit marked dilatation. Swollen mitochondria with peripherally placed and disintegrated cristae are shown. Note the vesiculation of the smooth endoplasmic reticulum.
229 spherical and relatively large. The mitochondrial cristae were predominantly vesicular. Small inclusion bodies and lipid droplets could be seen in the mitochondrial matrix. There were lipid droplets of various sizes dispersed in the cytoplasm (Figs. 1 and 2). The nucleus was spherical, polysomes and free ribosomes were abundant. In the zona reticularis the epithelial cells were small and had fine structural features, similar to those of the zona fasciculata. Lipid droplets were limited in number, the smooth endoplasmic reticulum was vesicular and abundant. Mitochondria were abundant and of irregular shape. The cristae were both of the tubular and of the vesicular variety. There were many lysosomes and lipofuscin granules (Fig. 3). In the adrenal cortex from Bz-treated rats, cells from fasciculata and reticularis zones exhibited a pattern of similar alterations. The cells showed a dense nucleus with large chromatin clumps marginated and attached to the internal nuclear membrane. Some mitochondria showed rarefaction or loss of cristae. Free ribosomes and polysomes were usually numerous. In some cells, the cytoplasm was scanty and completely filled with large lipid droplets that compressed the remaining organelles and deeply indented into the nucleus (Figs. 4 and 5). Most cells, however, exhibited variable degrees of lipid accumulation in the cytoplasm.
Fig. 5. Electron micrograph ( x 8800)from the reticularis zone of a benznidazole-treatedrat adrenal. This illustration shows degenerativechanges as evidenced by the presence of lipid droplets that empty the cytoplasmand disintegrate the internal architecture.
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Discussion Observations from this study provide evidence that Bz administration to rats induces ultrastructural alterations in the adrenal cortex. This finding adds weight to the evidence that some nitroheterocyclic compounds may have cortico-adrenotoxic properties as we anticipated in our studies with another nitroheterocyclic drug, the nitrofurane nifurtimox (Nfx) [19]. In the case of Bz, the alterations were also observed in the fasciculata/reticularis area, but they were different in nature from those caused by Nfx [19]. These alterations involve mitochondria and the smooth endoplasmic reticulum but to a lesser extent than in the case of Nfx [19]. In contrast to the case of Nfx, Bz alters the nucleus and the cytoplasm, the latter showing numerous lipid droplets of very large size. Nuclear alterations might be causally related to the previously reported ability of Bz reactive metabolites to covalently bind to DNA and different nuclear protein fractions [8]. Similarly, covalent binding of Bz reactive metabolites to phospholipids and proteins from mitochondria or smooth endoplasmic reticulum [3,5,10] may account for the alterations reported in these organelles, Nitroreductase activity associated with the formation of reactive moieties was observed in both, microsomes and mitochondria. Correlation between Bz nitroreductive biotransformation, production of reactive metabolites and toxic insult was repetitively demonstrated by our and other laboratories in different studies on mammals [2,3,7-12] and Trypanosoma cruzi [4]. Adrenal mitochondrial and microsomal Bz nitroreductase activity was oxygenbut not carbon monoxide-sensitive in microsomes or only slightly sensitive in mitochondria. This lack of sensitivity of carbon monoxide suggests lack or minor P-450 participation since carbon monoxide is known to block P-450 mediated biotransformations [13]. More likely, Bz would be reduced at the level of the flavinedependent P-450 reductase in adrenal microsomes [13,15,20] and at the adrenodoxin reductase level in adrenal mitochondria [13,15,20]. Adrenal cytosolic Bz nitroreductive biotransformation, in contrast to previous observations in other organs [3,5,11] was not observed. Marked lipid accumulation in adrenal cytoplasm of fasciculata and reticularis cells deserves detailed analysis. Several toxicants are known to produce abnormally large quantities of non-organelle associated lipids in the fasciculata/reticularis area, destroying cell structure and function [21,22]. The general mechanism of these effects is still not clear. Some lipidosisinducing agents are: clotrimazole [23], o-p-dichlorodiphenyldichloroethane [24], methanol [21], aniline [25] and aminoglutethimide [26]. At present we are not able to suggest a mechanism that accounts for the ability of Bz to lead to adrenal lipid accumulation. It is possible that the known inhibitory effects of Bz on P-450 dependent hydroxylations [10] could play a role. Steroid adrenal hydroxylations are known to be mediated by P-450 in microsomal and mitochondrial fractions [15,19,20]. Further studies are needed to test this hypothesis. Finally it could be of significance, to learn whether cortico-adrenal disfunction occurs in people during Bz clinical use since many thousand human beings are currently treated with this drug.
Acknowledgement This work was supported by a grant from the Secretaria de Ciencia y T~cnica (SECYT) Argentina.
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References 1 World Health Organization (WHO), Workshop on guidelines for standarized protocols for the chemotherapy of Chagas' disease. TDR/CHEMCHA/DC WKSHP/81.3 (1981), p. 1. 2 R. Docampo and S.N. Moreno, Biochemical toxicology of antiparasitic compounds used in the chemotherapy and cbemoprophylaxis of American Trypanosomiasis (Chagas' disease). Rev. Biochem. Pharmacol., 7 (1985) 159. 3 J.A. Castro and E.G.D. de Toranzo, Toxic effects of nifurtimox and benznidazole, two drugs used against American Trypanosomiasis (Chagas' disease). Biomed. Environ. Sci., 1 (1988) 19. 4 E.G.D. de Toranzo, J.A. Castro, B.M. Franke de Cazzulo and J.J. Cazzulo, Interaction of Benznidazole reactive metabolites with nuclear and kinetoplastic DNA, proteins and lipids from Trypanosoma cruzi. Experientia, 44 (1988) 880. 5 M. Masana, E.G.D. de Toranzo and J.A. Castro, Reductive metabolism and activation of benznidazole. Biochem. Pharmacol., 33 (1984) 1041. 6 R. Nagel and I. Nepomnaschy, Mutagenicity of 2-antichagasic drugs and their metabolic deactivation. Mutat. Res., 117 (1983) 237. 7 N.B. Gorla and J.A. Castro, Micronucleus formation in bone marrow of mice treated with nifurtimox and benznidazole. Toxicol. Lett., 25 (1985) 259. 8 N.B. Gorla, M.I. Diaz G6mez and J.A. Castro, Interaction ofbenznidazole reactive metabolites with rat liver deoxyribonucleic acid and nuclear proteins. Arch. Int. Pharmacodyn. Ther., 280 (1986) 22. 9 G.D. Castro and J.A. Castro, Studies on pentane evolution by rats treated with nifurtimox or benznidazole. Toxicology, 35 (1985) 3191. 10 M. Masana, E.G.D. de Toranzo, M. Rubio and J.A. Castro, Effect of benznidazole on the mixed function oxygenase system from rat liver microsomes. Arch. Int. Pharmacodyn. Ther., 276 (1985) 4. 11 A.S. Bernacchi, C.R. de Castro, E.G.D. de Toranzo and J.A. Castro, Effect of nifurtimox or benznidazole administration on rat testes: Ultrastructural observations and biochemical studies. Exp. Mol. Pathol., 45 (1986) 245. 12 C.R. de Castro, E.G.D. de Toranzo, A.S. Bernacchi, M. Carbone and J.A. Castro, Ultrastructural alterations in ovaries from Nifurtimox or Benznidazole-treated rats: Their relation to ovarian nitroreductive biotransformation of both drugs. Exp. Mol. Pathol., 50 (1989) 358-397. 13 R. Sato and T. Omura, Cytochrome P450, Academic Press, New York, 1978, p. 5. 14 J.A. Castro, M.I. Diaz G6mez, E.C. de Ferreyra, C.R. de Castro, N. D'Acosta and O.M. de Fenos, Carbon tetrachloride effect on rat liver and adrenals related to their mixed function oxygenase content, Biochem. Biophys. Res. Commun., 47 (1972) 315. 15 H.D. Colby and R.C. Rumbaugh, Adrenal Drug Metabolism, in T. Gram (Ed.). Extrahepatic metabolism of drugs and other foreign compounds, SP Medical and Scientific Books, New York, 1980, p. 239. 16 J.A. Luft, Improvements in epoxy resin embedding method. J. Biophys. Biochem. Cytol., 9 (1961) 409. 17 M.L. Watson, Staining of tissue sections for electron microscopy with heavy metals 11. Applications of solutions containing lead and barium. J. Biophys. Cytol., 4 (1958) 475. 18 J.H. Venable and R. Coggeshall, A simplified lead citrate stain for use in electron microscopy. J. Cell. Biol., 25 (1965). 19 C.R. de Castro, E.G.D. de Toranzo, M. Carbone and J.A. Castro, Ultrastructural effects of nifurtimox on rat adrenal cortex related to reductive biotransformation. Exptl. Mol. Pathol., 52 (1990) 98. 20 J. Blanck, G. Smettan and S. Greschner, The cytochrome P450 reaction mechanism. Kinetic aspects, in K. Ruckpane and H. Rein (Eds.), Cytochrome P450, Akademic-Verlag, Berlin, 1984, p. 120-128. 21 W.E. Ribelin, The effects of drugs and chemicals upon the structure of the adrenal gland. Fund. Appl. Toxicol., 4 (1984) 105. 22 H.D. Colby, Adrenal Gland Toxicity: Chemically induced dysfunction. J. Am. Coll. Toxicol., 7 (1988) 45. 23 D. Tettenborn, Toxicity of chlotrimazole. Postgrad. Med. J., 50 (1974) 17. 24 M.M. Hart, R.L. Reagan and R.H. Adamson, The effect of isomers of DDD on the ACTH-induced steroid output, histology and ultrastructure of the dog adrenal cortex. Toxicol. Appl. Pharmacol., 24 (1973) 101.
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K. Kovaks, J.A. Blascheck, E. Yeghiayan, S. Takeyame and C. Gardell, Adrenocortical lipid hyperplasia induced in rats by aniline: A hystologic and electron microscopy study. Am. J. Pathol., 62 (1971) 473. R.N. Dexter, L.M. Fishman, R.L. Ney and G.W. Liddle, Inhibition of adrenal corticosteroid synthesis by amino glutethimide. Studies on the mechanism of action. J. Clin. Endocrinol. Metab., 27 (1967) 473.
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Benznidazole-induced ultrastructural alterations in rat adrenal cortex. Mechanistic studies C.R. de Castro, E.G. Diaz de Toranzo and J.A. Castro Centro de Investigaeiones Toxieol6gieas (CE1TOX) CITEFA/CON1CET Zufriategui 4380. 1603 Villa Martelli, Prov. de Buenos Aires (Argentina)
(Received February 1lth, 1992; accepted June 10th, 1992)
Summary Benznidazole (Bz) (N-benzyl-2-nitro-l-imidazole acetamide) is a drug used against Chagas' disease, a parasitic disease afflicting several millions of Latin Americans. Bz administration to Sprague-Dawley male rats at 100 mg/kg p.o. caused subcellular alterations in the adrenal cortex involving fasciculata and reticularis zones but not in the glomerulosa. There is Bz nitroreductase activity in the adrenal microsomal and mitochondrial fractions but most of it is localized in mitochondria. Activity in the two fractions requires NADPH under anaerobic conditions. Mitochondrial Bz nitroreductase activity was inhibited by oxygen. A minor but statistically significant inhibition was observed in mixtures incubated under carbon monoxide. Microsomal Bz nitroreductase activity was not detected under oxygen atmosphere and was not inhibited under carbon monoxide. No Bz nitroreductase activity mediated by xanthine oxidase or aldehyde oxidase was detected in the cytosolic fraction from rat adrenals. Electron microscopic examination of the adrenal cortex from Bz-treated animals revealed cells with marked lipid accumulation and alterations in nuclei, endoplasmic reticulum and mitochondria in the reticularis and fasciculata zones. In vitro results suggest a Bz nitroreductive activation, with minor or null P-450 participation, leading to reactive metabolites able to cause damage in various organelles. Key words: Benznidazole; Fasciculata and Reticularis Zones; Adrenals; Chagas' disease
Introduction B e n z n i d a z o l e (Bz) is a d r u g p r e s e n t l y in use in the c h e m o t h e r a p y o f C h a g a s ' disease, a p a r a s i t i c disease a f f l i c t i n g several m i l l i o n p e o p l e in L a t i n A m e r i c a [ 1 - 3 ] . Bz t h e r a p y , h o w e v e r , has s i g n i f i c a n t u n d e s i r a b l e side effects w h i c h f r e q u e n t l y cause p a t i e n t s to s t o p t r e a t m e n t [2,3]. B o t h c h e m o t h e r a p e u t i c a n d toxic side effects o f Bz a p p e a r to be r e l a t e d to Bz r e d u c t i v e b i o t r a n s f o r m a t i o n [4-12]. In p r e v i o u s studies w e r e p o r t e d the o c c u r r e n c e o f u l t r a s t r u c t u r a l a l t e r a t i o n s in diff e r e n t rat tissues r e l a t e d to Bz n i t r o r e d u c t a s e a c t i v i t y [11,12]. Bz n i t r o r e d u c t a s e activity was p r e v i o u s l y r e p o r t e d to be a s s o c i a t e d w i t h the p r e s e n c e o f c y t o c h r o m e P-450 (P-450), P - 4 5 0 r e d u c t a s e , x a n t h i n e o x i d a s e a n d a l d e h y d e o x i d a s e [2,3,5,11,12]. Correspondence to: C.R. de Castro, Centro de Investigaciones Toxicol6gicas (CEITOX) CITEFA/CONICET. Zufriategui 4380, 1603 Villa Martelli, Prov. de Buenos Aires, Argentina.
0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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This work describes ultrastructural alterations in adrenal cortex from rats pretreated with Bz and their relation to the Bz nitroreductase activity in several cellular fractions. Materials and methods
Chemicals Bz (N-benzyl-2-nitro-l-imidazole acetamide) was a gift from Hoffman La Roche & Co. Ltd. All other chemicals used were of analytical grade. Animals and treatments Sprague-Dawley male rats (220-250 g) bred in our laboratory were used throughout these experiments. The animals were housed in wire cages. Temperature in the animal room was 23 + 2°C and the relative humidity was between 35 and 65%. Light was on from 06:00 h-18:00 h. Animals had access to food (Purina Chow rat diet) and water at all times during the experiments. Bz was given orally as a single dose of 100 mg/kg suspended in 1% carboxy methylcellulose (CMC). The control group received vehicle only. Adrenal glands from anesthetized animals dosed with 50 mg/kg body wt. pentobarbital were removed 24 h after Bz administration. The rats were then sacrificed without delay. Adrenals isolated from untreated animals bred under the same conditions were used for the in vitro studies. The rats were sacrificed and the adrenal glands rapidly excised and processed. Isolation of subcellular fractions Adrenal glands were homogenized in a Teflon-glass Potter-Elvehjem homogenizer with 10 vols. of 1.15% KC1. The homogenate was centrifuged for 10 min at 600 × g and after the pellet was resuspended in 10 vols. of 1.15% KC1 a further centrifugation under the same conditions followed. The resultant supernatants were collected and centrifuged for 15 min at 8000 × g. The pellet obtained was the mitochondrial preparation which was washed once with 20 vols. of 1.15% KC1 before use. The remaining supernatants were centrifuged again for 1 h at 105 000 × g in order to obtain the pelleted microsomes and the supernatant cytosol fraction. This fraction was dialyzed overnight in the cold room against 2 1 of 20 mM phosphate buffer (pH 7.4). All these operations were performed at 2-4°C. Enzymatic and chemical determinations All incubations were done in 20 ml septum-stoppered vials at 37 °C. In a final volume of 2.5 ml of 20 mM phosphate buffer (pH 7.4) vials contained: (a) the subcellular fraction (either a final concentration ranging from 0.49 to 1.40 mg protein/ml of mitochondria, or 0.25 to 0.64 mg protein/ml of microsomes, or 0.96 to 1.11 mg protein/ml of cytosol); (b) 0.05 ml NADPH-generating system composed by: 0.3 M Tris-HC1 buffer (pH 7.4), 0.2 ml; 1 M MgCI2, 0.2 ml; isocitric acid dehydrogenase type IV from porcine heart, 0.6 ml; dl-isocitric acid trisodium salt, 124 mg; N A D P H sodium salt, 20 rag; and (c) Bz to a final concentration 0.288 raM. For cytosol incubations N A D P H was omitted and either N-methylnicotinamide (2.5 mM) or hypoxanthine (0.25 mM) as substrate was included. Vials containing the
225
different subcellular fraction suspensions were bubbled with oxygen-free nitrogen, pure oxygen or carbon monoxide for 5 min prior to the addition of N A D P H and Bz as a dimethylformamide solution. The final concentration of dimethylformamide in the incubation mixture was 2%. After incubation for 30 or 60 min reactions were terminated by the addition of 1 ml of 15% zinc sulfate. Incubation mixtures were added to 2.5 g of NaC1 and extracted with 7.0 ml of ethylacetate. The organic phase was read in a spectrophotometer at 315 nm. At this wave length the drug has maximum absorption. Absorbance was compared against a calibration curve of different concentrations of Bz in ethyl acetate. Enzymatic activity was determined by measuring the decrease in absorbance due to the reduction of the Bz nitrogroup. Absorbance values at 30 or 60 min of incubation were subtracted from the absorbance value at zero incubation time taken as a blank. This procedure compensates for the absorbance of endogenous substances other than the substrate. Under these experimental conditions Bz nitroreductase activity was linear with time and protein content.
Electron microscopy Both adrenals from each rat (5 animals per group) were prepared for electron microscopy. Ten cubes per adrenal gland of approximately 1 mm were cut under 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), fixed by immersion for 24 h, and fixed in 1% osmium tetroxide. The cubes were immersion-stained in block with uranyl acetate, dehydrated in graded ethanol solutions and propylene oxide and later embedded in epoxy resine [16]. Thick sections for orientation purpose were obtained with a LKB ultratome using glass knives and stained with toluidine blue. Thin sections were cut with diamond knives, stained with uranyl acetate [17] and lead citrate [18] and examined in a Philips EM 300 electron microscope. Statistics A decision tree for selecting appropriate statistical procedures (parametric or nonparametric) was applied to results from every experiment [ 111. Significance between two groups was established in all cases using F-test followed by Student's t-test.
TABLE 1 BENZNIDAZOLE N I T R O R E D U C T A S E ACTIVITY IN D I F F E R E N T CELLULAR FRACTIONS FROM RAT ADRENALS a Values are the means ± S.D. of three different experiments. Each result was obtained by pooling the adrenal glands from the number of rats indicated in parentheses. Subcellular fraction
Bz nitroreductase activity nmol Bz/min per mg protein
Mitochondria Microsomes
2.775 ~- 0.200 (22) 1.338 ± 0.091 (22)
aThe different subcellular fractions were incubated at 37°C under a N 2 atmosphere with Bz and NADPH generating system for 30 min in case of mitochondria and for 60 min in case of microsomes.
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Results
Bz nitroreductase activity in different cellular fractions from rat adrenals Mitochondria and microsomes from adrenal glands exhibited Bz nitroreductase activity under anaerobic conditions and in the presence o f N A D P H (Table I). Most o f the activity was f o u n d in the mitochondrial fraction. W h e n the cytosolic fractions were incubated at 37°C for 60 min under a nitrogen atmosphere with Bz and either N-methyl-nicotinamide or hypoxanthine as substrate, xanthine oxydase (0.038 4- 0.03 nmol Bz/min per mg P) and aldehyde oxydase (0.026 ± 0.045 nmol Bz/min per mg P) activities were negligible.
Mitochondrial and microsomal Bz nitroreductase activity from rat adrenals under different atmospheres Mitochondrial Bz nitroreductase activity was almost completely abolished when incubated under oxygen atmosphere. A statistically significant inhibition was observed when incubated with pure c a r b o n monoxide. Microsomal Bz nitroreductase activity was not detected when the fraction was incubated under pure oxygen and no differences were observed between carbon monoxide and nitrogen incubations (Table II).
Bz-induced ultrastructural changes in adrenal cortex The glomerulosa, fasciculata and reticularis zones were examined and since Bzinduced damage was confined exclusively to the zones fasciculata and reticularis, description o f alterations will be confined to these two zones. The fasciculata cells from control rats were rich in smooth endoplasmic reticulum membranes and mitochondria. The smooth endoplasmic reticulum was vesicular and arranged in layers surrounding lipid droplets and mitochondria. Mitochondria were numerous,
TABLE II BENZNIDAZOLE NITROREDUCTASE ACTIVITY IN DIFFERENT CELLULAR FRACTIONS FROM RAT ADRENALS UNDER DIFFERENT ATMOSPHERES a Values are the means 4- S.D. of three different experiments. Each results was obtained by pooling the adrenal glands from the number of rats indicated in parentheses. Subcellular fraction
Atmosphere
Bz nitroreductase activity nmoles/min/mg protein
Mitochondria
Nz 02 CO
2.830 4- 0.033 (69) 0.026 4- 0.032b (69) 2.266 4- 0.191b (69)
Microsomes
N2 02 CO
1.126 + 0.590 (53) 0 b (53) 1.103 4- 0.419 (53)
alncubation mixtures were as in Table I except for the different atmospheres. bp < 0.05 compared with the N 2 atmosphere.
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Fig. 1. An electron micrograph ( × 8800) from the fasciculata zone of a control rat adrenal showing round nucleus, abundant spherical mitochondria, numerous free ribosomes and polysomes and well developed Golgi.
Fig. 2. Electron micrograph ( x 14 000) from the fasciculata zone of a control rat adrenal. In some cells, the cytoplasm exhibits numerous normal-looking mitochondria and a deep mvagination occupied by a lipid droplet.
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Fig. 3. Reticularis zone of a control rat adrenal showing a round nucleus, numerous mitochondria with lamellar and tubular cristae and the presence of lipofuscin granules in the cytoplasm ( x 8800),
Fig. 4. Electron micrograph ( x 14 000) from the fasciculata zone of a benznidazole-treated rat adrenal. The nuclear membrane and the Golgi exhibit marked dilatation. Swollen mitochondria with peripherally placed and disintegrated cristae are shown. Note the vesiculation of the smooth endoplasmic reticulum.
229 spherical and relatively large. The mitochondrial cristae were predominantly vesicular. Small inclusion bodies and lipid droplets could be seen in the mitochondrial matrix. There were lipid droplets of various sizes dispersed in the cytoplasm (Figs. 1 and 2). The nucleus was spherical, polysomes and free ribosomes were abundant. In the zona reticularis the epithelial cells were small and had fine structural features, similar to those of the zona fasciculata. Lipid droplets were limited in number, the smooth endoplasmic reticulum was vesicular and abundant. Mitochondria were abundant and of irregular shape. The cristae were both of the tubular and of the vesicular variety. There were many lysosomes and lipofuscin granules (Fig. 3). In the adrenal cortex from Bz-treated rats, cells from fasciculata and reticularis zones exhibited a pattern of similar alterations. The cells showed a dense nucleus with large chromatin clumps marginated and attached to the internal nuclear membrane. Some mitochondria showed rarefaction or loss of cristae. Free ribosomes and polysomes were usually numerous. In some cells, the cytoplasm was scanty and completely filled with large lipid droplets that compressed the remaining organelles and deeply indented into the nucleus (Figs. 4 and 5). Most cells, however, exhibited variable degrees of lipid accumulation in the cytoplasm.
Fig. 5. Electron micrograph ( x 8800)from the reticularis zone of a benznidazole-treatedrat adrenal. This illustration shows degenerativechanges as evidenced by the presence of lipid droplets that empty the cytoplasmand disintegrate the internal architecture.
230
Discussion Observations from this study provide evidence that Bz administration to rats induces ultrastructural alterations in the adrenal cortex. This finding adds weight to the evidence that some nitroheterocyclic compounds may have cortico-adrenotoxic properties as we anticipated in our studies with another nitroheterocyclic drug, the nitrofurane nifurtimox (Nfx) [19]. In the case of Bz, the alterations were also observed in the fasciculata/reticularis area, but they were different in nature from those caused by Nfx [19]. These alterations involve mitochondria and the smooth endoplasmic reticulum but to a lesser extent than in the case of Nfx [19]. In contrast to the case of Nfx, Bz alters the nucleus and the cytoplasm, the latter showing numerous lipid droplets of very large size. Nuclear alterations might be causally related to the previously reported ability of Bz reactive metabolites to covalently bind to DNA and different nuclear protein fractions [8]. Similarly, covalent binding of Bz reactive metabolites to phospholipids and proteins from mitochondria or smooth endoplasmic reticulum [3,5,10] may account for the alterations reported in these organelles, Nitroreductase activity associated with the formation of reactive moieties was observed in both, microsomes and mitochondria. Correlation between Bz nitroreductive biotransformation, production of reactive metabolites and toxic insult was repetitively demonstrated by our and other laboratories in different studies on mammals [2,3,7-12] and Trypanosoma cruzi [4]. Adrenal mitochondrial and microsomal Bz nitroreductase activity was oxygenbut not carbon monoxide-sensitive in microsomes or only slightly sensitive in mitochondria. This lack of sensitivity of carbon monoxide suggests lack or minor P-450 participation since carbon monoxide is known to block P-450 mediated biotransformations [13]. More likely, Bz would be reduced at the level of the flavinedependent P-450 reductase in adrenal microsomes [13,15,20] and at the adrenodoxin reductase level in adrenal mitochondria [13,15,20]. Adrenal cytosolic Bz nitroreductive biotransformation, in contrast to previous observations in other organs [3,5,11] was not observed. Marked lipid accumulation in adrenal cytoplasm of fasciculata and reticularis cells deserves detailed analysis. Several toxicants are known to produce abnormally large quantities of non-organelle associated lipids in the fasciculata/reticularis area, destroying cell structure and function [21,22]. The general mechanism of these effects is still not clear. Some lipidosisinducing agents are: clotrimazole [23], o-p-dichlorodiphenyldichloroethane [24], methanol [21], aniline [25] and aminoglutethimide [26]. At present we are not able to suggest a mechanism that accounts for the ability of Bz to lead to adrenal lipid accumulation. It is possible that the known inhibitory effects of Bz on P-450 dependent hydroxylations [10] could play a role. Steroid adrenal hydroxylations are known to be mediated by P-450 in microsomal and mitochondrial fractions [15,19,20]. Further studies are needed to test this hypothesis. Finally it could be of significance, to learn whether cortico-adrenal disfunction occurs in people during Bz clinical use since many thousand human beings are currently treated with this drug.
Acknowledgement This work was supported by a grant from the Secretaria de Ciencia y T~cnica (SECYT) Argentina.
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