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JUOEH20(1)!11-19 (1998) 11 [Original)
Pathological Study Splenomegaly Associated with Cadmium-inducedof Anemia in Rats
Tetsuo HAMADA', Akihide TANIMOT02, Nobuyuki ARIMA!, Yoshihiro IDEL,
Takakazu SASAGURI2, Shohei SHIMiVIRI2, Yoshitaka MURATA',
Ke-Yong WANG2 and Yasuyuki SASAGURIL'
'Dopartment of'Surgical Pathotogy, University llosPital ?Dopartment qf Palhology and Cell Biotogy, Scheol of' Adlrdicine, (1itib'ersity of Occapational and Environmental Health., .lapan. }'dhatanishi-ku, Kiialp,ztshu 807-8imr, .1apan
Ahstract : Splenomegaly was observcd both in male and R)malc Spraguc-Dawley rats after 1 week of exposure to CdC12 (O,6 mg (:d/kg/day), Sp]een weight reached about double that in controls by 8 weeks of Cd exposure.
Histopathologica] cxamination of the enlargcd spleen rcvcaled that iron- and lipid-laden histiocytes were clustered in tha periarterial lymphatic sheath, and the red pulp appeared to be expanded. It is noteworthy that electron microscopy rcvealed markcd poikilocytosis and Hcinz body formation in red blood cells (RBCs) in both the sinus and cord, Histiocytes werc swollen by a granu}ar substance in the cytoplasm and also many secondary lysosomes.
Thesc morphological findings indicatc that degradation of damagcd RBCs
induced by exposure to Cd might bc promoted in the splccn and possibly cause splenomcgaly, This RBC damage-hcmolysis-splcnomegaly sequence
is also considered to be associated with the etioiogy of Cd-induced anemia,
In addition to the abnormal RBC degradation, nuclel of ]ymphocytes in thc
Cd-cxposcd spleen exhibited high elcctron density, consistent with a
preapoptotic statc suggesting the immunosupprcssive effhct ofCd.
Kay woralf :spienomegaly, poiki]ocytosis, Heinz body, cadmiurn, anemia,
(Received 18 November 1997, accepted I9January 1998)
Introduction
Cd was reported to cause anemia in dogs as early as 1896 [1], and subsequently, anemia was found in humans after Cd exposure [2]. A later study indicated that anemia appearcd to be onc of the carliest signs of Cd poisoning in higher vertebrates [3]. The main characteristic of Cd-induced ancmia is the presence of microcytic, hypochromic red blood cells (RBCs),
possibly resulting from iron deficicncy [4] . However, other investigators havc
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12 THApt･IADA et al
suggested that hemolysis or bone marrow suppression may be the cause of the anemia [5, 6] ; thus, the pathogenesis of Cd-induccd anemia is still a subject of debate. NVe noticcd that in rats given subcutancous (sc) irijections ofCd, splenomegaly developed during thc early stages of administration in association with anemia
onsct. Aithough exposure to Cd has been reportcd to induce splenomegaly in mice [7, 8] and rats [9, 10], to our knowlcdgc there have bcen no reports describing the histopathological as well as ultrastructural features ofsplenomegaly in relation to Cd-induced anemia. In this study, we focused on patholc)gic changes in the spleen of rats with subchronic Cd intoxication and assessed the
significance ol' splenomegaly in relation to thc ctiology of Cd-induced ancmia.
Materials and Methods Animats Ninety-six Sprague-Dawlcy rats (Kyudo,Japan), IO or 1 1 weeks ofage, were given a conventional tbod Oapan CLEA, CE2) and tap water ad libitum. Rats in the exposure group were administered O.6 mg Cd/kg/day as CdC12 solution rlihc (about 1/100 LD50) subcutaneously lbr 5 days a week fbr up to 8 wecks, same volume ofphysiological saline was injected into control rats, Body weights
of the rats were measured ()nce a week.
HistqPathotogical examination
Six rats including 4 in the exposure group and 2 in the control group were sacrificed every week from 1 to 8 weeks during the experimental period under general anesthesia by inhalation of cthyl ether, and the spleens were removed immcdiately. The rcsected spleens were weighed, photographed, hemisected
sagittally across the splenic hilus, and fixed in 15% fbrmalin solution, Then, paraflin sections (3 ptm thick) were stained with hematoxylin and eosin (H&E), Berlin blue, Turnbull blue and periodic acid-Shiff (PAS). Oii red O staining was using frozen sections of tissue after immersion pcrfbrmcd 5 ,um formalin-fixed in gum-sucrose solution overnight,
Electron microscopic examination Small tissuc blocks cut from the resected spleen, about 1 × 1 × 1 mm in sizc, were fixed with cacodylate-buffered (O.l M, pH 7.4-) glutaraldehyde solution (2.5%) for 2 h and postfixecl with cacodylate-buflered osmium tetroxide (1.0%) for 1 h. After embedding in Epon 812 (TAAB, I.ondon, UK), ultrathin sections wcre cut, stained with uranyl acetate and lcad citratc, and examined using an electron microscope (1200CX, JEOL, Tokyo, Japan),
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Splenomegaly Induccd by Cadmiurn 13
Results Animals
There was no s{gnificant difference in body weight change between Cd- exposed and control rats during the administration period (not shown). Splenomegaly developed and was significant (P Macroscopic examination The spleens excised from these rats exposed to Cd fbr more than 1 week were enlarged with an incrcased consistency and rough surface. The capsules ofthese "sugar-icing" splcens were distended, and showed a appearance (Fig. 2), Histopathological examination H&E sections of the enlarged spleen exposed to Ccl revealed an expansion of the red pulp and clustered histiocytes with brown granules in their cytoplasm in the periarterial lymphatic sheath (Fig. 3nl). These brown granules showcd positive staining with Berlin blue, indicating iron deposition in the cytoplasm (Fig. 3-2). Oil red O staining also demonstrated lipid deposits corresponding to the Berlin blue-positive granules (data not shown). 2.2 o---N;ngBcoo)･.--N-oec 2.0 1.8 1.4 1.0 O 2 4 6 8 Administration period(VVeeks) Fig. 1. Relative spleen to body w'eight (SW/BW) ratio of' cadmium exposed rats, Relative SivVIBI'V ratio meaiis (cadniium exposed spleenlbody'wcight)f twicc that of control spleen in 8 w Cd expnsure. NII-Electronic Library Service The UOEHAssociationUOEH Association ofofHealthSclences Health Sciences 14 T H"ADA et a( Fig 2llepresentahNe rriac rosLopic dcatures ot control dnd Ccl cxposcd spleens The spLeen exposed Lo O 6 mg CdClplKg {sL) foi 8" showcd marked cnlargerncnt, thc capgulc havmg a rough surfacc and suqar icing appearance (× 2O, Bar = 1O crn) es Fig 3 Histopatholoqical changes in Cd cxpesecl splecn rhere aie gome ciustcred histiocNtes (arroM) scattercd in thc pemartctial lymphatic sheath in caclmium exposed spleLn (1, H&h hectien) l"hLsc hisnoqtes arc posiuve foi iron staming == = {2, Beilin blue stdining) CBdr 1 100,rim, Bar 2 5UIim) NII-Electronic Library Service The UOEHAssociationUOEH Association ofofHealth Health Sciences SpLcnomegaly Induced b}, Cadmiutn I5 Electron mieroscopic examination Ultrastructural examination of the enlarged spleen revealed the diffuse presence of RBCs with a defbrmed and irregular contour in the splcnic tissue, particularly in thc dilated cords, Some of the dcformcd RBCs were fbund to be reticulocytes, recognizable by their small number of organelles (Fig. 4-1), These damaged RBCs contained granular or angular inclusions with high electron density in both the cord and sinus. The electron-densc inclusions were compatible with Heinz bodies [11, 12] . Some electron-dcnse globules in thc histiocytes were fbund to be consistent with phagocytosed RBC fragmcnts (Fig. 4-2), Thcre were many secondary lysosomes and membranous or g'ranular substances in the cytoplasm ofswollen histiocytes. A noteworthy feature was the presence o{' glycogenosomes engulfing a largc amount of glycogcn in the autophagic vacuolcs [13] . I.ymphocytes showed mild morphoEogical alterations, fbr example, a split fbrmation between the inner and outer nuclear membranes, and higher nuclear electron density in comparison with control specimens (Fig. 4m3). wwww ` ee , ,/X,ma ,/ ,,ee.k bu.,x Fig. 4. Poikilocytosis in caclmium-exposcd gplccn. (l) Elcctron micrograph demonstrates that /Kacanthocyte tear drop or bizarrc RBCs are in sinii$ or clilated cords of as(A), Cl') CB) present Ladmium-exposecl spleen (Bar = 5 ,am). Hcinz bodics in ad-exposed splecn. (2) Granular to anbuular Heinz bedies (arrow) arc noticcd in RBCs in cadmium-exposed splcen. Somc HcinL bodics arc attachcd to RBC mcmbrane and c)thers are floatlng in RBC cytoplasm Onseit). (Bar = 5,ttm) Ultrastructural alterdtions in the histiocytes and 1>･rriphocyLes in Cd-exposed splecn. (3) In Cd-cxposcd splccn, a large gl>,cogenosemc (G), various heLeiophasFosomes and incmbranous substances are found m the histiocytes, High eLectron density in nucleoso]ne and == cli]atat{on of nuc/lcar rnembrane (ariow) are noted m the Iyrnphocytcs (Bar 2ptmm). Discussion Therc havc reports of studics dcscri the splenomegaly inbccnducedseveral by Cd. Earlyexperimentalstudies showed that Cdbinghad an NII-Electronic Library Service The UOEHAssociationUOEH Association ofofHealth Health Sciences 'l' 16 I'EAMADA et at immunosuppressive efi'ect on mice and induccd splcnome.ffaly [7, 14] . Later, changes in essential metals after administration ofCd were analyzed biochernically in the enlargcd spleen and other organs such as the atrophic thymus and pancreas [8, 9, 15]. A recent study has indicated that Cd atalow dose can cause anemia by increasing the RBC density and accclerating RBC sequestration, presumably in the spleen [10] , However, there have been no morpholegical investigations of the enlarged splccn induced by Cd in relation to the etiology of anemia. In the present study, clustered sidcrophages were evident histologically in the periarterial lymphatic shcath, suggesting acceieration ofhemolysis in the enlarged spleen. Ultrastructural examination of RBCs demonstratcd that poikilocytosis could be induced by Cd. Poikilocytesis is a state ofincreased abnormality ofRBC shape, including acanthocytes, spur cells or fragmented RBCs that are known to be present in various human diseases associated with anemia. Many chemical agents have been rcportcd to induce hemolytic anemia, but poikilocytosis is induced by only a few oxidative drugs [16] . To date, there has been one report rlihis indicating that poikilocytosis develops in Cd inLoxication [17]. report pointed out that anemia was charactcrized by significant reticulocytosis, anisocytosis, poikilocytosis, iron deficiency and marked alterations in thc antioxidant and metabolic status of RBCs, Although the etiology of poikilocytosis in Cd intoxication is unknown, we speculate that Iipid peroxidation ofthe RBC membrane may be rcsponsible for the deformity, since Cd has been reported to exert a lipicl peroxidation effect [I8] , Peroxidativc stress on RBC has be.en shown to induce hemolysis in abetalipoproteinemia [19]. Cd also altcrs thc surfacc ncgative charge of the RBC membrane [20]. It is notcworthy that in the present study Heinz bodies were found to be fbrmed in Cd-exposed RBCs. Hcinz bodies result from an oxidative irijury that leads to denaturation and precipitation of hemoglobin, particularly abnormal or unstable hcmoglobin and hcmoglobin in RBC with glucose-6-phosphate dehydrogenase deficiency [21]. Heinz bodies are known te be generatcd in vitro by oxidative stressors such as phenylhydrazine through reduction of the erythrocyte anion transport [22] or changes in the cytoskeletal components of the RBC membrane [23] . Heinz body formation in Cd intoxication may support the hypothesis that Cd influences the antioxidant state in RBC. Some heavy metals including zinc [24] and coppcr [25] rcportcdly cause hcmolytic anemia with Heinz body formation in dogs and shecp, rcspcctivcly, Howcvcr, to our knowlcdge, there have been no reports indicating' that Cd induces Heinz body fbrmatic)n in RBC, Heinz body formation is considered to accelerate the hemolytic process because RBCs-containing Heinz body are removcd from Lhc circulation predominantly by the splecn [26]. NII-Electronic Library Service The UOEHAssociationUOEH Association ofofHealth Health Sciences Splenorncgaly Inducttd by Cadmium 17 Therefbre, it is reasonable to assume that Cd aflbcts the stability of the RBC membrane, resulting in poikilocytosis and subsequent promotion ofhemolysis in the spleen. This sequence ofhemolysis is a likely cause ofsplenornegaly and also a factor operating in anemia, in addition to other possiblc mechanisms involved in Cd intoxication. It is not known why a transient incrcase of spleen weight occurred only in female rats in 1 w. Acute response to the early dose ofCd and sex difference of rats may be associated with the phenomenon. Glycogenosome formation in Ccl intoxication was first discovered by Sato et al, in rat sciatic nerves [27] . Iiater, Cd was found to have a strong inhibitory eflbct on neutral a-glucosidase, resulting in formations of abnormal glycogen macromolecules [28] , It is highly probable that glyco.aen mctabolism in splcnic histiocytes is affected by Cd. The hi.crher nuclear electron density ofCd-exposed lymphocytes in comparison with control preparations might represent a since vitro and in vivo cxperimental studies preapoptoticphenomenon [29], both in have demonstrated that apoptosis is inducible by Cd [30, 31, 32]. This study was partly supported by a UOEH research grant fbr promotion of occupational health. References 1. Athanasiu M & Langloi P (1896): Comparison ofsalts ofcadrnium ancl zinc. Arch Physiol 28: 251-263 2. Friberg L (1950): Health hazards in the manufacture of alkaline accurnulators with special reference to chronic cadrnium poisoning. Acta Med Scand 240 (suppl): IL-124･ 3. Nomiyama K, Sugata Y, Yamamoto A & Nomiyama H (1975): Efi'ects ofdietary cadmiuin on '1'oxicol rabbits. I. Early signs of cadmium intoxication. Appl Pharmacol 31: 4-12 4. Huebers HA, Huebers E, Csiba E, Rummel W & Finch CA (1987): The cadmium effect on iron absorption. Am J CIin Nutr 45: 1007-1012 5, Johansson-S.]'obeck M-L & Larsson A (1978): The eflect ofcadrnium on the liematology arid on the activity of delta-aminolevulinic acid dehydratase (AJ,A-D) in blood and hematopoictic tissucs of the flounder, Pleuronectcs flcsus I.. Environ Res 17: 191-204 6. Hays Ei" & Margaretten N (1985): Iiong-term oral caclmium produces bone marrow hypop[asia in mice. Exp Hematol 13: 229-234 7, Boze]ka BE, Burkholdcr PM & Chang LW (1978): Cadmium, a metallic inhibitor ofantibody- rnediated immunity in mice. Environ Res 17: 390-402 8. Suzuki KT, Yamada YK & Shimizu F (1981): E$sential metals and mctallothionein in cadmiutn-induced thyrnic atrophy and splenornegaly. Biochem Pharmacol 30: 1217-1222 9. Suzuki KT, Ohnuki R, Yaguchi K & Yamada YK (1983): Acc:umulation and chemical fbrms of cadmium and its cffect on essential metals in rat spleen and pancreas, J Texicoi Enviren Health 11: 727-737 le. Kunimoto M & Miura T (1986): Density increment and decreased survival of rat recl bloocl cells induccd bv cadmium. Environ Res 39: 86-95 ' 11. Heinz R (1890): Morphologische Veranderungen der roten Blutcorperchen durch Git'te. Virchows Arch Pathol Anat Physiol Klin Med 122: li2-116 NII-Electronic Library Service The UOEHAssociationUOEH Association ofofHealth Health Sciences 18 THAM.'XDA et al - 12 Rilkind RA & Danon D (1965): Heinz body anemia an ultrastructuraE study, II, Rcd ccll scqucstration and destruction. Blood 26: 433-448 13 Iwamasa T, Tsuru T, Sasaki M, Hamada T & Takeuchi T (I980): Ultrastructural and biochcmical studics ofg}ycogenosome lbrrnation in ascites hepatoma AHI3 cells. Pathol Res Pract 168: 173-184･ 14･ Kojima A & Tamura SI (1981): Acute eifects ofcadmiurn on delayed-type hypersensitivity in mice, Jpn J Med Sci Biol 34: 281-291 15 Maitani T & Suzuki KT (1982): Changcs of cssential metal levels in selected tissues and splenomegaly induced by the hziection of suspcnding cadmium salt into mice, Toxicol Appl Pharrnacol 62: 219-227 16. Wickrarnansinghe SN(l986): Non-immune acquired hemolytic anemia. In: Systemic Pathology. Vel 2 Blood and Bone Marrow. 3rd ed. 27, Sato K, Iwamasa T, Tsuru T & Takcuchi T (1976): An ultrastructural study of chronic cadmium chloride-induced neuropathy, Acta Neuropathol Berl 41: 185-190 28, IIamada T, Iwamasa T, Tsuru T, Sato K & Takcuch{ T (1981): Mechanisin ofglycogenosome fbrmation in axons of cadmium-induced neuropathy. Neurotoxicology 2: 33-4･1 29. Cohcn GM, Sun XM, Snowdcn RT, Ormcrod MG & Dinsdalc D (1993): ldcntification ofa transitional preapoptotic population of thymocytes. J Immullol 151: 566-574 30, Hamada T, Nakano S, Iwai S, Tanimoto A, Ariyoshi K & Koide O (l991): Pathological study on beagles at'ter long-term oral adrninistration ol' cadrnium, Toxicol Pathol 19: 138-147 'I' 31, Tanimoto A, Hamada & Koide O (1993)[ Cell death and regeneration of renal proximal tubular cells in rats with subchronic cadmium intoxication. Texicol Pathol 21: 341-352 32. Hamada T, Sasaguri T, Tanimoto A, Arima N, Shimajiri S, Abc T & Sasaguri Y (1996)i NII-Electronic Library Service The UOEH AssooiationAssociation of Health SciencesSoienoes SplenomegaLy Indueed by Gadlnium ［9 − Apoptosis of human kidney 293 cclls is promoted by polymerizcd cadmium meta ］lothionein， − Biochcm Biophys Rcs Comrnun 219： 829 834 カ ド ミ ウ ム 誘発 貧血 症 ラ ッ トに お け る 脾 腫の 病理 学的研 究 1 2 2 ： 2 濱 田 哲 夫 ， 谷 本 昭英 ， 有 馬 信 之 ， 井 出 良浩 ，笹 栗 毅 和 正 Z 2 王 ビ 之 2 島尻 平 ， 村 田 義 隆 ， 克舗 ， 笹栗 靖 1産 業医科大学 病院 病院病理 部 2 産業 医科大学 医学部 第 二 病理 学教室 一・ 要 旨 ： 貧 血 は カ ド ミ ウ ム （Cd ）中毒 に お け る 最早 期 徴 候 の つ と し て よ く知 ら れ て い る ． 我 々 は 貧血 に 伴 っ て 認 め られ る 脾腫 と変 形赤 血 球 （PC ）増 多症 に 注 目 し ， Cd 誘 発 貧 血 症 の 発症機構 に つ い て 検討 を加 え た ． ラ ッ トで は 脾腫 は Cd の 慢性投 ｛1 に よ り， 1 週 間後 に は 明 らか と な り，そ の 重量 は 8 週 間 で は 対照 の 約 2 倍 と な っ た ．腫大脾 の 電 子 顕 微鏡的検索 で は ハ イ ン ツ 小 体 （IIB）の 形成 と著 明 な PC が 目 立 っ た． HB の 形 ・ 成 は 赤血 球 の 脾 内溶 rill．を ，次 い で PC 増 多症 を 促進 す る こ と が 知 ら れ て い る ． 方 ， 赤血 球 で は 膜 の 脂 質過酸化 が PC の ，ヘ モ グ ロ ビ ン の 酸化 的変性 が HB の 原 因 と され い こ の こ つ い で こ て る． れ ら と か ら ， Cd は 初 め に 赤 エ血球 膜 異常 PC 増 多症 を ひ き起 し， こ の 異常赤血 球が脾 で 処理 され る た め脾 腫 が 招 来 され る こ とが 示 され た ．こ の よう 一 一 一 に 赤ikL球異常 脾 内溶血 脾腫 の 連関 は ， Cd 誘発貧 血 発症 の メ カ ニ ズ ム の つ と し て 重 要 で あ る ． − JUOEH （産業 医大誌 〉，20 〔1 ）： 11 19 （1998） 一 NII-ElectronicN 工 工 Eleotronio Library Service