Bull. Environ. Contain. Toxicol. (1991) 47:288-295 9 1991 Springer-Vedag New York Inc. Environmental ~C o n t a m i n a t i o n | a n d Toxicology Similar Effects In Vivo of Two Aluminum Salts on the Liver, Kidney, Bone, and Brain of Rattus norvegicus Ajoy Kumar Roy,* Geeta Talukder, and Archana Sharma Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Calcutta 700 019, India The widespread distribution of aluminum (AI) compounds in nature and their use have stimulated considerable interest in the toxicity of this metal. Aluminum accumulation has been suggested to be an associated phenomenon in various human diseases such as renal dialysis dementia, senile dementia, dialysis osteomalacia, microcytic hypochromic anaemia, gastrointestinal toxicity and Alzheimer's disease (for detail see Ganrot 1988, Krishnan et al 1988). The daily intake of AI has been estimated to be 9-14 mg (Pennington 1988). Pharmacological dose of A1 as antacids is estimated to be l-3g daily (Greger and Baler 1983). Following absorption, the metal is widely distributed throughout human body (Venugopal and Luckey 1978, Skalsky and Carchman 1983, Ganrot 1986) and accumulates in different tissues. The present work was undertaken to observe the effects of different concentrations of aluminum following oral for various durations on various organs of ingestion rats and also to compare two different A1 salts at doses having the same amount of AI. The findings can be of relevance owing to the widespread use of aluminum compounds by oral route either as medicines or unintentionally through utensils and cookwares. MATERIALS AND METHODS One hundered thirty five laboratory bred, healthy albino male rats Rattus ~ about 2 months old, weighing 120-150g were used for the experiment. The animals were maintained on standard pellet diet (Hindustan Lever, India) and given water ad libitum. Send reprint request to Prof, A.Sharma at above address * Present address: Plant Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi-284003, India. 288 five animals were kept l a b o r a t o r y conditions. in one cage under s t a n d a r d Two salts of a l u m i n u m [aluminum sulphate, A I 2 ( S O d ) s . 1 8 H20 and p o t a s s i u m a l u m i n u m sulphate KAI(S04)2. 12H20] were used and d i s s o l v e d in d e i o n i z e d water. The following d o s e s (Table i) were a d m i n i s t e r e d d a i l y up to 21 days to the animals by garage. Table 1 s h o w i n g d i f f e r e n t d o e s e s of test chemical Salt Aluminum sulphate Dose in terms of LD~o i/5th I/lOth 1/20th 1/30th 1/40th 1/50th Amount of salt/lOOg body wt. LDao " " " " " Potassium aluminum sulphate 212.0 i08.0 53.0 35.5 26.5 21.2 mg mg mg mg mg mg 76.5 mg 50.3 mg Amount of metal/lOOg body wt. 17.18 8.59 4.29 2.86 2.18 1.72 mg mg mg mg mg mg 4.29 mg 2.86 mg Control sets were maintained by feeding the animals with deionized water. For each c o n c e n t r a t i o n in each experiment and for controls, 15 rats ~ere used. Five rats were s a c r i f i c e d at the end of each week. The tissues namely, heart, liver, kidney, brain, testes, stomach and femur bone were d i s s e c t e d out and fixed in p h o s p h a t e - b u f f e r e d formalin. Fixed tissues were processed for microtomy, sections were p r o c e s s e d and stained f o l l o w i n g the h a e m a t o x y l i n - e o s i n y d o u b l e s t a i n i n g s c h e d u l e (Pearse 1988). RESULTS AND D I S C U S S I O N The effects of the two salts were similar at the comparable doses. The general effects are d e s c r i b e d below. Dose dependent c y t o t o x i c effects were o b s e r v e d in the liver. C o n t r o l sets s h o w e d normal s t r u o t u r e (Fig. IA). The lower d o s e s 1.72,2.16 and 2.88 mg of AIS04 and 2.86 mg of KAIS04 affected the periphery of the lobule 289 showing cytoplasmic degeneration and the nucleus as seen by hyperchromacia. With increasing doses multifocal degeneration of the entire liver tissue followed by the fibrous tissue proliferation was observed (Fig. IB). T h e r e was also overall increase in c o n g e s t i o n and d i l a t a t i o n of the sinusoids. The effects of both salts were similar at c o m p a r a b l e doses. Treatment with the lowest dose (1.72 mg) of AIS04 showed a slight swelling of the tubules of the cortex,but otherwise tissue s t r u c t u r e was n o r m a l . W i t h increasing dose (2.16 mg) increased swelling and degeneration of the cortical tubules were seen (Fig. IC) .Treatment with 2.86 mg of b o t h salts caused contraction of glomeruli and d e g e n e r a t i o n of d i s t a l tubules.Subcapsular necrosis in some areas with d i l a t a t i o n and d e g e n e r a t i o n of m e d u l l a was also n o t i c e d with i n c r e a s i n g period of t r e a t m e n t . A higher dose (4.29 mg) of both salts induced more marked change in the cortical tissue affecting glomeruli as well as tubules.Higher dose (8,59 mg) induced h a e m o r r h a g e and dilatation of tubules following prolonged exposure.Highest dose (17.18 mg) caused marked degeneration of tubules with loss of s t r u c t u r e (Fig. ID).Thus kidney also showed dose d e p e n d e n t d e g e n e r a t i o n primarily of the tubules followed by n e c r o s i s m o s t l y observed in the cortex. In higher d o s e s the m e d u l l a showed m a r k e d d e g e n e r a t i o n and cystic d i l a t a t i o n of the tubules. Treatment with 1.72 mg for 21 days and 2,16 mg for 7 days did not show any a p p r e c i a b l e c h a n g e . A f t e r 14 and 21 days there was d e g e n e r a t i o n of the n e r v e cells with dilatation of blood cells (figs. 2 A , 2 B ) . T r e a t m e n t with 2.86 and 4.29 mg of either salt caused neuronal degeneration of the c e r e b r a l c o r t e x w i t h d i l a t a t i o n of blood vessels which increased with duration of e x p o s u r e . W i t h higher d o s e s (8,59 mg and 17.18 mg) the d a m a g e of the nerve fibre was marked in the s u b c o r t i c a l region and in the base of the b r a i n . T h i s d e g e n e r a t i o n was multifocal (Figs.2C,2D) and visible without specific nerve stain. The effects were dose and duration dependent and similar in both salts at the doses compared. Testes did not show any h i s t o l o g i c a l d a m a g e up to 4.29 mg set of either salt.There was some evidence of s p e r m a t o g o n i a l cell d e c r e a s e after 21 days of treatment with 8.59 mg and 14 days with 17.18 mg a l u m i n u m sulphate. Heart did not show any h i s t o l o g i c a l effect d o s e s tried in this study. 290 in all the PLATE 1 : Microphotographs of section through liver (figs.iA-B),kidney (figs.iC-D) showing. Fig. IA : normal structure in control rats (CaxlO0) Fig. IB : multifocal degeneration of liver cells localized around the portal vessel following treatment with aluminum sulphate (8.59mg/lOOg body wt.) for 21 days (Ca *I00) Fig, IC : swelling and degeneration of cortical tubules following treatment with aluminum sulphate (2.88 mg/lOOg body wt.)for 21 days (Ca*lO0) Fig, ID : marked degeneration of tubules with loss of structure following treatment with aluminum sulphate (17.18 mg/lOOg body wt.) for 14 days (Ca*800) 291 PLATE 2 : Microphotographs of section through brain (fig~, 2A-D)showing : Fig. 2A : slight degeneration of nerve cells and dilatation of blood vessels following treatment with aluminum sulphate (2.86 mg/lOOg body wt.) for 14 days (Ca *iO0) Fig. 2B : Fig. 2A magnified (Ca *600) Fig. ZC : multifocal degeneration and marked fibrosis following treatment with aluminum sulphate (17.18 mg/lOOg body wt,)for 21 days (Ca *I00) Fig. 2D : Fig.2C magnified (Ca *600) 292 Bone was not affected in control and up to 2.86 mg treated sets of both salts. In the 4.29 mg treated set of both salts there was multifooal degeneration and decalcification which increased with increasing duration,however,the obteoblast cells remained apparently normal.Degeneration of calcified bone and irregularity of osteoblasts was observed markedly in animals treated with 6.59 and 17.16 mg of aluminum sulphate. Mucosal layer was thickened with some alteration of superficial layer. Hyperplasia and ulceration of stomach were recorded in some regions in 8.59 mg and 17.16 mg treated sets. Lower doses of both salts up to 4.29 mg were not toxic. Aluminum has been found to be neurotoxic (See Roy 1987,Krishnan e t a l . 1988.Lipman et al.1988).It induces neurofibrillary degeneration and neuronal loss. The present observations indicate that the effects of A1 on brain tissues depend both on the dose administered and the duration of treatment. Liver possibly plays a significant role in the metabolism o f Ai.Previous in v ire and in vitro studies have indicated the toxic responses of the hepatic tissue to A1 (Ebina et ai.1984) as well as its accumulation in liver (Yokel 1983,Alfrey et al.1985,Klein et al 1988).Rats fed with 257 and 1075 ug Al/g diet for 67 days accumulated significantly greater amount of AI in their tibias,kidney and liver (Greger et al. 1986)while rats injected intraperitoneally for i0 days with 2.7 mg showed significant A1 accumulation in brain, liver, spleen,bone and heart (Burnatowska-Hledin and Mayor 1964). The observations made here confirm previous reports of damage induced by treatment with A1 on brain and liver.These effects increase with increasing doses and longer periods of exposure.Similar damage was induced in kidney as well.The degenerative effect on the liver is followed by fibrosis observed after prolonged treatment in two highest doses of AISO~.Similarly the kidney cortex also shows tubular degeneration.The effects are suggestive of functional damage on a prolonged treatment.The deposition of A1 in the brain neuronal fibers has been reported in Alzheimer's disease or pro-senile dementia(Perl and Brody 1980) although the correlation is not yet conclusively established.The observation that prolonged use causes increase of fibrosis of the cerebral cortex following neuronal degeneration can be correlated with the toxic effect of AI. 293 The decrease of osteoid cells and degeneration of calcified bones is suggestive of osteomalacic changes as observed in dialysis syndrome (Hodsman et ai.1982, Goodman 1986). However,despite the earlier records of accumulation of A1 in skeleton,testes (Venugopal and Luckey 1978)and heart and other muscles (Skalsky and Carchman 1983) the observations made here do not show any appreciable histological damage in these organs. The gastro-intestinal tract in particular,is not affected to any significant extent,despite prolonged oral administration. There is no significant difference in the effects of the two salts, when given in concentrations which would release equivalent amount of the metal per lOOg body weight (see Table l).This suggests that the two salts are metabolized equally effectively in the animal system. Acknowledgments : The authors are grateful to Professor A.K. Sharma, Programme Co-ordinator, for the laboratory facilities provided and University Grants Commission, New Delhi for the financial assistance. 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