Introduction

The heavy metal cadmium (Cd++) is an industrial andenvironmental pollutant (Harstad and Klaassen, 2002). Itis commonly used in environmental studies because it is

highly toxic (Faroon et al., 1994). Cd exposure leads topathological effects in the liver (Friedman and Gesek,1994), testes (Shen and Sangiah, 1995), brain, nervoussystem (Provias et al., 1994), kidney (Novelli et al.,1999), spleen and bone marrow (Yamano et al., 1998).

Turk J Zool29 (2005) 283-289 © TÜB‹TAK

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The Histochemical and Ultrastructural Effects ofCadmium on Mouse EDL Muscle

Fatime GEY‹KO⁄LUDepartment of Biology, Faculty of Arts and Sciences, Atatürk University, 25240, Erzurum - TURKEY

E-mail: geyikogluff@yahoo.com

Aysel TEMELL‹Department of Biology, Education Faculty of Kâz›m Karabekir, Atatürk University, 25240, Erzurum - TURKEY

Özgen VURALERDivision of Histology and Embryology, Medical Faculty, Atatürk University, 25240, Erzurum - TURKEY

Hasan TÜRKEZDepartment of Biology, Faculty of Arts and Sciences, Atatürk University, 25240, Erzurum - TURKEY

Received: 20.09.2004

Abstract: The histochemical and ultrastructural effects of cadmium (Cd) on the extensor digitorum longus (EDL) muscles of malemice were investigated. Two doses of Cd (0.1 and 1.0 x 10-4 M) were injected into the mice. The animals were killed at the end of48 h and their EDL muscles excised. Three types of fibers (I, IIA and IIB) were described by the succinic dehydrogenase (SDH) methodin the control groups. The mitochondrial and sarcotubular contents of the fibers were different.

The histochemical and ultrastructural characteristics of the EDL muscles in mice given Cd did not differ from those of the controls.Therefore, the results of this application are not given separately. However, the high dose of Cd (1.0 x 10-4 M) caused an increasein the SDH activity in all types of muscle fibers. In addition, ultrastructural alterations of the sarcoplasmic reticulum andmitochondria, a reduction in energetic reserves (lipid droplets, glycogen) (types I and IIA) and myofibrillar degenerations weredetected. Grain-like deposits were also observed in the cytoplasm of type IIB fibers.

Key Words: Cadmium, EDL muscle, Mice, SDH, Ultrastructure, Cytopathology.

Fare EDL Kas› Üzerine Kadmiyumun Histokimyasal ve Ultrastrüktürel Etkileri

Özet: Erkek farelerin extensor digitorum longus (EDL) kaslar› üzerine kadmiyum (Cd)’un histokimyasal ve ultrastrüktürel etkileriaraflt›r›ld›. Farelere Cd’un iki dozu (0,1 ve 1,0 x 10-4 M) enjekte edildi. Hayvanlar 48 saatin sonunda öldürüldü ve onlar›n EDL kaslar›ç›kar›ld›. Kontrol gruplar›nda süksinik dehidrojenaz (SDH) metodu ile liflerin üç tipi (I, IIA ve IIB) tan›mland›. Liflerin mitokondri vesarkotübüler içerikleri farkl›yd›.

Cd verilen farelerde EDL kaslar›n›n histokimyasal ve ultrastrüktürel özellikleri kontrollerdekinden farkl› de¤ildi. Bu yüzden, buuygulama sonuçlar› ayr›ca verilmemektedir. Ancak, kadmiyumun yüksek dozu (1,0 x 10-4 M) kas liflerinin tüm tiplerinde SDHaktivitesi üzerine art›fla neden oldu. Ayr›ca, sarkoplazmik retikulumun ve mitokondrilerin ultrastrüktürel de¤ifliklikleri, enerjirezervlerinde azalma (lipid damlalar›, glikojen) (I ve IIA tipleri) ve miyofibriller dejenerasyonlar tesbit edildi. Tip IIB liflerininsitoplazmas›nda tane benzeri tortular gözlendi.

Anahtar Sözcükler: Kadmiyum, EDL kas›, Fare, SDH, Ultrastrüktür, Sitopatoloji.

Among the various effects induced by Cd in biologicalsystems, oxidative damage to membrane lipids byperoxidation, a phenomenon termed lipid peroxidation(LPO), has been reported in the liver and kidneys (Xu etal., 2003). For longer exposure times, Cd inducedchanges in α-actin disorganization in the smooth musclecells (Azou et al., 2002). There were distinct alterationsof the intercalated disk structures of the cardiac muscle inrats dependent upon the level and time of exposure(Kolakowski et al., 1983).

Three types of fibers were distinguished in theextensor digitorium longus (EDL) muscle of mice byhistochemical reactions (types I, IIA and IIB) (Freitas etal., 2002). Succinic dehydrogenase (SDH) revealedsignificant activity in subgroup IIA, very little activity insubgroup IIB, and intense activity in group I. SDH is anenzyme of mitochondria. The enzymes (gill ATPases,brain acetylcholinesterase, liver glutamate oxaloacetateand glutamate pyruvate transaminases) may act asreliable biomarkers of Cd toxicity (Torre et al., 2000). Tothe best of our knowledge, histochemical andultrastructural studies on the EDL muscle of mice exposedto Cd have not been reported. Therefore, histochemical(SDH activity in mitochondria) and ultrastructuralanalyses were performed to examine whether cadmiumchloride (CdCl2) has a dose-dependent effect on EDLmuscle.

Materials and Methods

Twenty adult male BALB/c mice obtained from theVeterinary and Animal Diseases Research Laboratorywere used. The mice were housed in standard plasticcages (22 ± 2 °C, 65% humidity, artificial lights from06.00 to 20.00). The mice were reared on a standarddry diet and water. For the experiments, the mice weredivided into 4 groups (5 mice/group), 2 of which receivedsterile saline (control) while the other 2 were exposed toCdCl2. The mice were randomly assigned to 1 of 2treatment groups. Daily doses of 0.1 x 10-4 M(subchronic dose) and 1.0 x 10-4 M (chronic dose) ofCdCl2/kg were given by subcutaneous injections for 2days to the different groups (Satoh et al., 1982). Themice were anesthetized with sodium pentobarbital (40-70 mg/kg). The anesthetized mice were sacrificed bycervical dislocation. All procedures were carried out inaccordance with the “Guiding Principles in the Use ofToxicology’’ of the Society of Toxicology (Regunathan etal., 2002).

The mice were weighed. The EDL muscle was excised.In the first procedure, the muscle was frozen (Freitas etal., 2002). Cross sections (12 µm) were taken along themuscle at -20 °C an a cryostat (Eddinger et al., 1985).The sections were stained with SDH to determine themitochondrial activity in the muscle fibers (Bancroft andStevens, 1982; Geyiko¤lu et al., 2002). In the secondprocedure, a small portion of EDL was removed from thecenter of the muscle length, oriented on a wooden stick,and fixed in phosphate-buffered (0.1 M, pH 7.2-7.3)glutaraldehyde (3%)-formaldehyde for 3 h, with mincingafter 30 min. The tissues were washed with phosphate-buffered (0.1 M, pH 7.2-7.3) sucrose (0.15 M), post-fixed in buffered osmium (1%), dehydrated in ethanol,rinsed in propylene oxide and embedded in Epon-Araldite.Thin sections were obtained on a Reichert OmU2ultramicrotome, stained with saturated aqueous uranylacetate (5%) and lead citrate (4%), and examined undera Siemens Elmiskop I electron microscope (Bancroft andStevens, 1982).

The results were expressed as means ± S.E. A pairedStudent’s t-test was used for comparing values betweenthe experimental groups.

Results

The average body weight of the control groups was22.0 ± 1 g (mean ± SE). The weight was not changed byCdCl2 exposure.

The type IIA fibers of the control EDL showed thinnerand rarer subsarcolemmal/intermyofibrillar deposits offormazan than type I fibers in the SDH reaction. On theother hand, type IIB fibers typically showed weakreactivity, depicted by rare formazan precipitate (Figure1).

The intensity of the SDH reaction in all fibers did notvary with exposure to 0.1 x 10-4 M of CdCl2. Therefore,the figures of those receivig a subchronic dose are notpresented. However, all of fibers exhibited dose-dependent increased reactions after treatment with 1.0 x10-4 M of CdCl2 (Figure 2). It was also observed that theangular fibers (IIB) were interspersed between the otherfibers after exposure to Cd.

In the control EDL, the transmission electronmicroscope (TEM) studies elucidated ultrastructuraldifferences in the distribution of the mitochondria of the

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3 types of fibers. The subsarcolemmal mitochondria werenumerous in the type I, intermediate in type IIA, butalmost absent in type IIB. Type IIB fibers had moredeveloped sarcoplasmic reticulum (Figures 3A, B, C).

No pathologic effects were observed after theapplication of CdCl2. However, compared with EDL cellsfrom the control, the main structural alterations observedin fibers after exposure to 1.0 x 10-4 M of CdCl2 were anincrease in the number of subsarcolemmal mitochondria,which were more prominent in types IIA and IIB (Figures4A, B); changes in the mitochondria (destroyedmitochondria and long, branched mitochondria (Figures4B, 5A); individual concentric whorls of sarcoplasmicreticulum cisternae in the muscle fibers (Figure 5B); anddegeneration of myofibrils in type I fibers (Figure 5C).

The number of lipid droplets of EDL muscle in thecontrol mice tended to be abundant in the type I fibers,and intermediate in type IIA fibers. However, glycogen

particles in type I were scarce compared with those ontype IIA (Figures 6A, B).

In application of 1.0 x 10-4 M of CdCl2, theultrastructural changes observed in the fibers were anabsence of lipid (in type I fibers), a reduction in thenumber of lipid droplets (in type IIA) and an absence ofglycogen (in type I and IIA fibers) (Figures 7A, B). Inaddition to these alterations, grain-like electrondensedeposits in the cytosol of some type IIB fibers (Figure 8)were observed.

Discussion

According to the results obtained from investigationson different animal species, organic chemicals and metalscan influence the tissue glycogen content (Srivastava,1982; Segner and Braunbeck, 1998). In the presentstudy, an absence or reduction of glycogen and lipid

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Figure 1. Cross section of the EDL from control mice. Type I (arrow),type IIA (asterisk) and type IIB (double arrow). SDH, x680.

Figure 2. Increased reactions in cross section of the EDL. Type I(arrow), type IIA (asterisk), type IIB (double arrow),angular fiber (curved arrow). SDH, x680.

droplets was a general feature in the EDL of the miceafter exposure to 1.0 x 10-4 M of CdCl2. Our studyindicates enhanced utilization of energetic reserves in EDLmuscle. Therfore, it seems reasonable to suggest that thetoxic effects of Cd on mammals are similar to thoseobserved in starvation, since CdCl2 could induce loss ofglycogen and lipids. In mammals that underwent foodrestriction over a 24-48 h period, tissue glycogen wasspent and lipolysis was enhanced to spare the remainingcarbohydrate reserves and peripheral proteins (Herzbergand Farrell, 2003). Again, in this study, no differenceswere observed in the body weights of control and Cd-exposed mice. Thus, animals that survived Cd exposuremay show a metabolic shift and a compensatorydevelopment for maintenance of weight.

On the other hand, dose levels of Cd stimulateddevelopmental and electrophysiological effects (Papp etal., 2003). Furthermore, the rate of protein synthesiswas reduced by cadmium acetate in mouse trachea organculture (Lag et al., 1986). In our study, the degenerationof myofibrils was seen due to the effect of 1.0 x 10-4 Mof CdCl2. It was suggested that the increased energydemand might have led to an immediate stimulation ofprotein catabolism, which resulted in a degeneration ofmyofibrils in type I fibers. The observed ultrastructuralalterations on the sarcoplasmic reticulum were alsoreported in the granular endoplasmic reticulum ofdifferent cell types after exposure to metals (Pawert etal., 1996). Ghadially (1999) presented 2 views regardingthe nature of concentric membranous bodies of granular

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Figures 3. Electron micrographs showing subsarcolemmal mitochondria (double arrow) in the longitudinal sections of thecontrol EDL. A) Type I has numerous mitochondria, B) Type IIA has a moderate number, C) In type IIB mitochondriaare scarce but sarcoplasmic reticulum is numerous (arrow). x6000.

endoplasmic reticulum: 1: they represent a degenerativechange or an elaborate autophagic vacuole; and 2: theyrepresent a regenerative change leading to a specializedtype of hypertrophy of the endoplasmic reticulum thatmay have functional significance. The concentricallyarranged sarcoplasmic reticulum cisternae observed in thepresent study were found accompanying normal cisternaein the muscle cell; artificial cisternae do not seem to have

occurrred. Therefore, these were probably related toincreased function due to glycolysis by 1.0 x 10-4 M ofCdCl2. We thought that the increases in SDH activity inthe EDL muscle of mice exposed to a high dose of Cdcould also reflect altered mitochondrial function throughincreased glycolysis and lipolysis. We also interpreted thissituation due to mitochondrial alterations under anelectron microscope. Thus, mitochondrial changes can beconsidered related to adaptation of the EDL to themetabolism of the exogenous agent. In fact, it has beenreported that mitochondrial function was altered by Cd(Al-Nasser, 2000; Kim et al., 2003). In the present study,ultrastructurally, the presence of inclusion bodies in thecytosol stands out. Previous studies established that asmall quantity of Cd is found in the nucleus andmitochondria, and the greater part is cytosol, where Cd is

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Figures 4. Electron micrographs showing subsarcolemmalmitochondria (double arrow) and destroyed mitochondria(arrow) in the longitudinal sections of EDL. A) Type IIA hasnumerous mitochondria, B) Type IIB has a moderatenumber. x6000.

Figures 5. Electron micrographs showing structural alterations of EDL.A) Long (double arrow), branched mitochondria (arrow),x20,000, B) Concentrically arranged sarcoplasmic reticulumcisternae (arrowhead), x50,000, C) Degeneration ofmyofibrils in type I fibers (curved arrow). x30,000.

bound to metallothionein (Topashka-Ancheva et al.,2003).

In conclusion, the degeneration of myofibrils, thedetected changes in mitochondria and sarcoplasmicreticulum, and the decreases of glycogen and lipid in thesarcoplasm of EDL strongly support the toxicologicalsignificance of increased doses of CdCl2.

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Figures 6. Electron micrographs showing lipid droplets (L) and glycogen (arrow) in thelongitudinal sections of control EDL. A) Type I. Lipid droplets are numerous andlarge, but glycogen is scarce, B) Type IIA. Lipid droplets (L) and glycogen (arrow) aremoderate in number. x20,000.

Figures 7. Electron micrographs of the longitudinal sections of EDL, inapplication of 1.0 x 10-4 M of CdCl2. A) Type I. Lipiddroplets and glycogen are lacking, B) Type IIA. Note thereduction in the number of lipid droplets (arrow) and theabsence of glycogen. x20,000.

Figures 8. Electron micrograph showing electrodense deposits(arrows) in the longitudinal sections of type IIB. x15,000.

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References

Al-Nasser, I.A. 2000. Cadmium hepatotoxicity and alterations of themitochondrial function. J. Toxicol. Clin. Toxicol. 38: 407-413.

Azou, B.L., Dubus, I., Ohayon-Courtes, C., Labouyrie, J.P., Perez, L.,Pouvreau, C., Juvet, L. and Cambar, J. 2002. Cadmium inducesdirect morphological changes in mesangial cell culture. Toxicol.179 (3): 233-245.

Bancroft, S.D. and Stevens, A. 1982. Theory and Practice of HistologicalTechniques. Churchill Livingstone, (2nd ed.) Edinburgh, p. 662.

Eddinger, T.J., Moss, R.L. and Cassens, R.G. 1985. Fiber number andtype composition in extensor digitorium longus, soleus, anddiaphragm muscles with aging in Fisher 344 rats. J. Histochem.Cytochem. 33 (10): 1033-1041.

Faroon, O.M., Williams, M. and O’Connor, R. 1994. A review of thecarcinogenecity of chemicals most frequently found at nationalpriorities list sites. Toxicol. Ind. Health. 10: 203-230.

Freitas, E.M. S., Silva, M.D.P. and Cruz-Höfling, M.A. 2002.Histochemical differences in the responses of predominantly fast-twitch glycolytic muscle and slow-twitch oxidative muscle toveratrine. Toxicon. 40: 1471-1481.

Friedman, P.A. and Gesek, F.A. 1994. Cadmium uptake by kidney distalconvolute tubule cells. Toxicol. Appl. Pharmacol. 128: 257-263.

Geyiko¤lu, F., Temelli, A. and Özkaral, A. 2002. Morphologicaladaptation of rat skeletal muscle to a cold environment. Turk. J.Vet. Anim. Sci. 26: 1121-1126.

Ghadially, F.N. 1999. As you like it, Part 2: A critique and historicalreview of the electron microscopy literature. Ultrastruct. Pathol.23: 1-17.

Harstad, E.B. and Klaassen, C.D. 2002. Gadolinium chloridepretreatment prevents cadmium chloride- induced liver damage inboth wild-type and MT-null mice. Toxicol. Appl. Pharmacol. 180:178-185.

Herzberg, G.R. and Farrell, B. 2003. Fasting-induced, selective loss offatty acids from muscle triacylglycerols. Nutr. Res. 23 (2): 205-213.

Kim, S.C., Cho, M.K. and Kim, S.G. 2003. Cadmium-induced non-apoptotic cell death mediated by oxidative stress under thecondition of sulfhydryl deficiency. Toxicol. Lett. 144 (3): 325-336.

Kolakowski, J., Baranski, B. and Opalska, B. 1983. Effect of long-terminhalation exposure to cadmium oxide fumes on cardiac muscleultrastructure in rats. Toxicol. Lett. 19 (3): 273-278.

Lag, M., Helgeland, K., Olsen, I. and Jonsen, J. 1986. Effects ofcadmium acetate and sodium selenite on mucociliary functions andadenosine triphosphate content in mouse trachea organ cultures.Toxicol. 39 (3): 323-332.

Novelli, E.L.B., Lopes, A.M., Rodrigues, A.S.E., Novelli Filho, J.L.V.B.and Ribas, B.O. 1999. Superoxide radical and nephrotoxic effectof cadmium exposure. Int. J. Environ. Health Res. 9: 109-116.

Papp, A., Nagymajtenyi, L. and Desi, I. 2003. A study onelectrophysiological effects of subchronic cadmium treatment inrats. Environ. Toxicol. Pharmacol. 13 (3): 181-186.

Pawert, M., Triebskorn, R., Graff, S., Berkus, M., Schulz, J. and Köhler,H.R. 1996. Cellular alterations in collembolan midgut cells as amarker of heavy metal exposure: ultrastructure and intracellularmetal distribution. Sci. Total. Environ. 181: 187-200.

Provias, J.P., Ackerley, C.A., Smith, C. and Becker, L.E. 1994. Cadmiumencephalopathy: a report with elemental analysis and pathologicalfindings. Acta Neuropathol. 88: 583-586.

Regunathan, A., Cerny, E.A., Villareal, J. and Bhattacharyya, M.H.2002. Role of fos and src in cadmium-induced decreases in bonemineral content in mice. Toxicol. Appl. Pharm. 185: 25-40.

Satoh, E., Asai, F., Itoh, K., Nishimura, M. and Urakawa, N. 1982.Mechanism of cadmium-induced blockade of neuromusculartransmission. Eur. J. Pharmacol. 77 (4): 251-257.

Segner, M. and Braunbeck, T. 1998. Cellular Response Profile toChemical Stress. Wiley, Spektrum Akademischer, Verlag, pp. 521-569.

Shen, Y.M. and Sangiah, S. 1995. Na+, K+, ATPase, glutathione andhydroxyl free radicals in cadmium chloride induced testiculartoxicity in mice. Arch. Environ. Contam. Toxicol. 29: 174-179.

Srivastava, D.K. 1982. Comparative effects of copper, cadmium andmercury on tissue glycogen of the catfish, Heteropneustes fossilis(Bloch). Toxicol. Lett. 11 (1- 2): 135-139.

Topashka-Ancheva, M., Metcheva, R. and Teodorova, S. 2003.Bioaccumulation and damaging action of polymetal industrial duston laboratory mice Mus musculus alba II. disturbances. Environ.Res. 92 (2): 152-160.

Torre, F.L., Salibian, A. and Ferrari, L. 2000. Biomarkers assessment injuvenile Cyprinus carpio exposed to waterborne cadmium.Environ. Pollut. 109: 277-282.

Xu, J., Maki, D. and Stapleton, S.R. 2003. Mediation of cadmium-induced oxidative damage and glucose-6-phosphatedehydrogenase expression through glutathione depletion. J.Biochem. Mol. Toxicol. 17 (2): 67-75.

Yamano, T., Shimizu, M. and Noda, T. 1998. Comparative effects ofrepeated administration of cadmium on kidney, spleen, thymus,and bone marrow in 2, 4 and 8 month old male Wistar rats.Toxicol. Sci. 46: 393-402.