Indian Journal of Experimental Biology Vol. 42, September 2004, pp. 876-883 Effectiveness of ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) against cerium toxicity Sadhana Shrivastava & R Mathur* School of Studies in Zoology, Jiwaji University, Gwalior 474011 , India Received 8 August 2003. Revised 12 May 2004 Therapeutic efficacy of EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid) against cerium intoxicated mice was studied. Administration of cerium showed significant decrease in haemoglobin percentage, RBC counts and blood glucose level with an increase in the activity of serum transaminases and WBC counts. Decrease in the activity of alkaline phosphatase and glycogen content was noted in liver and kidney after cerium exposure. Light and electron microscopical investigations showed that these changes were recouped considerably with the administration of EGTA suggesting its therapeutic efficacy against cerium toxicity. Keywords: Cerium, EGTA, Toxicity, Male mice IPC Code: 1m CI 7 A61 P Cerium is one of the 15 metals of lanthanide group of elements, which are very similar in their physical and chemical characteristics. The increased industrial use of cerium includes glass polishing, carbon arcs, as cracking agents and catalysts, camera lenses, television tubes, mirrors, medicine and other glass products I. It is also used in nuclear fission processes and is reported to cause skin lesions, acute chemical pneumonitis, bronchitis, bronchiotitis, focal hypertrophic emphysema and granulomatous peritonitis 1.2. The liver damage caused by a single intravenous injection of cerium is manifested by fatty liver 3 , morphological changes both in the liver mitochondria 4 and endoplasmic reticulum 5 and inhibitory effect on liver microsomal drug metabolizing enzymes 6 • It may also be responsible for in sufficient glycogen formation, which in its turn causes hypoglycemia 7 . It causes effects similar to those caused by carbon tetrachloride 8 accompanied by low blood sugar level. It also damages kidney9. A number of therapeutic agents have been reported as chelating agents against lanthanides viz ethylene diamine tetraacetic acid (EDT A), nitrilo triacetic acid (NTA), alpha tocopherol and phenobarbital 10-14. On the basis of the structural formula ethylene glycol bis (2-aminoethyl ether) tetra acetic acid (EGTA) seems *Correspondent author Phone:0751-50 16750 Fax:0751-2341450 Email: [email protected]to be a prospective chelator having oxygen with double bonds and CH 2 COOH groups. Therefore, the present study has been undertaken to evaluate the response of EGTA against cerium induced toxicity. Materials and Methods Cerous sulphate (Glaxo Laboratory Chemical Division, Glaxo Laboratory Ltd., Bombay, India) and ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) (Aldrich Chemical Company, Wisconsin, USA) were used. Other analytical grade laboratory reagents were procured from Merck (Germany) and Glaxo Chemicals (India). Male albino mice weighing 25-30 g were selected from departmental colony and kept under uniform conditions of light and temperature. The mice were kept on a standard pellete diet (Nav Maharashtra Chakan Veg Oil Ltd, New Delhi, India having metal contents in ppm dry weight Cu 10; Mn 33; Zn 45; Co 5) and drinking water ad libitum. The selected animals were divided into 4 groups. Groups 1 and 3 served as normal control and EGTA per se respectively. Groups 2 and 4 were experimental. All the groups were further divided into sub groups of 5 animals each. Chelating agent (EGTA) dose was prepared daily and pH (6.4) was adjusted with sodium bicarbonate before administration. Group1: Animals were given normal saline at a dose of 4ml kg·lday"1 sc for 7 days. Group 2: Animals were given cerous sulphate 0.5ml (lmM) day"1 sc for 7 days.
Transcript
Indian Journal of Experimental Biology Vol. 42, September 2004, pp. 876-883
Effectiveness of ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) against cerium toxicity
Sadhana Shrivastava & R Mathur* School of Studies in Zoology, Jiwaji University, Gwalior 474011 , India
Received 8 August 2003. Revised 12 May 2004
Therapeutic efficacy of EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid) against cerium intoxicated mice was studied. Administration of cerium showed significant decrease in haemoglobin percentage, RBC counts and blood glucose level with an increase in the activity of serum transaminases and WBC counts. Decrease in the activity of alkaline phosphatase and glycogen content was noted in liver and kidney after cerium exposure. Light and electron microscopical investigations showed that these changes were recouped considerably with the administration of EGTA suggesting its therapeutic efficacy against cerium toxicity.
Keywords: Cerium, EGTA, Toxicity, Male mice
IPC Code: 1m CI7 A61 P
Cerium is one of the 15 metals of lanthanide group of elements, which are very similar in their physical and chemical characteristics. The increased industrial use of cerium includes glass polishing, carbon arcs, as cracking agents and catalysts, camera lenses, television tubes, mirrors, medicine and other glass products I. It is also used in nuclear fission processes and is reported to cause skin lesions, acute chemical pneumonitis, bronchitis, bronchiotitis, focal hypertrophic emphysema and granulomatous peritonitis 1.2. The liver damage caused by a single intravenous injection of cerium is manifested by fatty liver3
, morphological changes both in the liver mitochondria4 and endoplasmic reticulum5 and inhibitory effect on liver microsomal drug metabolizing enzymes6
• It may also be responsible for in sufficient glycogen formation, which in its turn causes hypoglycemia 7. It causes effects similar to those caused by carbon tetrachloride 8 accompanied by low blood sugar level. It also damages kidney9. A number of therapeutic agents have been reported as chelating agents against lanthanides viz ethylene diamine tetraacetic acid (EDT A), nitrilo triacetic acid (NTA), alpha tocopherol and phenobarbital 10-14. On the basis of the structural formula ethylene glycol bis (2-aminoethyl ether) tetra acetic acid (EGT A) seems
to be a prospective chelator having oxygen with double bonds and CH2 COOH groups. Therefore, the present study has been undertaken to evaluate the response of EGT A against cerium induced toxicity.
Materials and Methods Cerous sulphate (Glaxo Laboratory Chemical
Division, Glaxo Laboratory Ltd., Bombay, India) and ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) (Aldrich Chemical Company, Wisconsin, USA) were used. Other analytical grade laboratory reagents were procured from Merck (Germany) and Glaxo Chemicals (India).
Male albino mice weighing 25-30 g were selected from departmental colony and kept under uniform conditions of light and temperature. The mice were kept on a standard pellete diet (Nav Maharashtra Chakan Veg Oil Ltd, New Delhi, India having metal contents in ppm dry weight Cu 10; Mn 33; Zn 45; Co 5) and drinking water ad libitum. The selected animals were divided into 4 groups. Groups 1 and 3 served as normal control and EGTA per se respectively. Groups 2 and 4 were experimental. All the groups were further divided into sub groups of 5 animals each. Chelating agent (EGTA) dose was prepared daily and pH (6.4) was adjusted with sodium bicarbonate before administration. Group1: Animals were given normal saline at a dose of
4ml kg·lday"1 sc for 7 days. Group 2: Animals were given cerous sulphate 0.5ml
(lmM) day"1 sc for 7 days.
SHRIV ASTA V A & MATHUR: EFFECTIVENESS OF EGT A AGAINST CERIUM TOXICITY 877
Group 3: Animals were given normal saline for 7 days followed immediately by EGT A at a dose of 5 mM day" I im for 5 days.
Group 4: Animals were given cerous sulphate as in group 2 followed immediately by EGT A as in group 3.
Animals of all the groups were sacrificed following day 7, 14, 21, 28 and 60 under light ether anesthesia. Blood, liver and kidney were processed for biochemical and histopathological studies.
Blood was collected directly by puncturing the retro-orbital venous sinus 15 for the haemoglobin percentage, RBC and WBC count I6
.17
, blood glucose level '8, AST and AL T19. Standard techniques were used to determine the glycogen content20 in fresh tissue. Isotonic buffered homogenate was prepared for the assay of proteins21 , acid and alkaline phosphatases22 and succinic dehydrogenase23 .
Liver and kidney were dissected out and washed with saline and fixed in Bouin's fluid, embedded in paraffin, sliced at 6 !lm and stained with haemotoxylin and eosin for examination by light microscopy. For electron microscopy tissues were fixed in 1 % osmium tetraoxide and processed for grid preparation and then studied at EM facitity of All India Institute of Medical Sciences, New Delhi, India.
The data obtained were analyzed using Student' s '(' test and one way analysis of variance (ANOYA) 24.
Results and Discussion Haematological parameters revealed severe
alterations and dose dependent changes after cerium per se treatment and values recouped significantly with EGT A treatment (Table 1). Cerium produced lysis of RBC, which consequently resulted in haemolytic anemia. It may be due to its action upon erythropoietic organs25.26. Ghosh et al. 7 reported reduced activity of certain erythrocyte membrane bound enzymes after lanthanide treatment. Increase in the number of lymphocytes after cerium treatment may indicate increase in defense mechanism against toxic effects of cerium.
In the present study, activity of AST and ALT increased significantly while blood glucose level was redUCed after cerium exposure. This rise in tram:,rninases may lead to tissue lysosomal disruption, phagocytosis and acute cellular injury 27,28. Lanthanides bind to serum proteins especially globulin and amino acids, and are transported to various body organs29. These parameters depicted improvement after EGT A treatment in cerium exposed mice. These changes were
maximum after 14 and 21 days durations (Table 1). Glycogen content depleted significantly after cerium per se exposure whereas fall in protein content was insignificant and the values were towards normal after treatment with EGT A (Table 2). Decreased blood glucose level may be due to hepatic injury and loss of glycogen5, 30. Arvela '4 and Malik et al. 31 have reported significant decrease in the activities of glucose-6-phosphatase, dehydrogenase, glutathione reductase, glutathione peroxidase and catalase in the epithelial cells of liver and kidney after lanthanide exposure.
Activity of acid and alkaline phosphatase showed sharp decrease after cerium per se exposure, however the values were restored after EGT A treatment (Table 3). The capacity of the lanthanides to form insoluble complexes with phosphatase enables them to function at low concentration as non-specific "phosphatases", ceasing off phosphate groups from A TP, glycerophosphate, nucleotides and nucleic acid32. The elevation may be associated with the damage of the cellular membrane, which may be responsible for the lysosomal stimulation through gross alteration in ionic balance and osmotic pressure leading to metabolic failure. Alteration in the activities of <x-amylase, lactate dehydrogenase, acid phosphatase and alkaline phosphatase in human serum have been reported after lanthanide exposure33. Ultrastructural changes in liver and kidney depicted injurious effects with loss of smooth endoplasmic reticulum and presence of cytoplasmic vacuoles as a result of cerous sulphate induced toxicity5,'4,27. Damaged organelles observed in the present study may be the result of cerous sulphate toxicity, which may induce the Iysosomes to release hydrolytic enzymes. In kidney, alkaline phosphatase originates from brush border and is involved in the transphosphorylation reaction and mediates the membrane transport27 . In the present study it is possible that the necrosis of the tubular epithelium along with several other changes as revealed in electronmicroscopic may have been responsible for reduced alkaline phosphatase acti vi ty .
EGT A administration improved the cerium induced depletion in activity of succinic dehydrogenase (Table 4). Succinic dehydrogenase a flavoprotein, found in mitochondria of liver of rat showed evidence of uncoupling of oxidative phosphorylation34. Administration of manganese is refsorted to cause inhibition in the SDH activity in liver 5. This is largely confirmed in the present study on the basis of electronmicroscopic study, which reveals reduction in
Table l--lnfluence of EGTA on haematological parameters of cerium exposed mice
(Values are mean ± SE from 5 observations in each group]
Groups Duration after Hb% RBCCount WBCCount ESR(mrnIhr) Hematocrit (%) Blood glucose treatment (g/looml) (million/mm) (thousand/mm3
) (mg/looml) (days)
Normal control 14.72±.73 9.70±A8 7.82±.39 0.15±.01 43A0±2.73 106.20±7A8 Cerium 7 12.80±.68 8.50±.52 8.20±.44 0.24±.01" 55.0Q±2.86" 87.44±5.44
One way ANOV A: F variance at 5 % level (treatments)
7 43.1* 14 42.1 * 21 44.4* 28 14.7* 60 1.41
P<0.05 "vs normal control, bvS cerium per se, * significant
the number of mitochondria, which tells upon the health of the tissues.
Liver is an organ responsible for major metabolic processes regulating functioning of many other organs/system and may lead to secondary effects on other organs. Cerium exposed mice had lymphocytic infiltration, hypertrophy in hepatocytes, cytoplasmic vacuolation and stumpy Kupffer cells (Fig. 1). With EGT A treatment chord arrangement was maintained. Binucleated cells and mitotic figures showed restoration (Fig. 2). Cerium exposure leads to fatty degeneration, focal necrosis and multinucleated giant cells in liver3.6. 13.36. Ultrastructure of liver showed enlarged mitochondria (Fig. 3) and dilatation of endoplasmic reticulum after cerium exposure (Fig. 4). Magnusson5 reported increased level of neutral fats from extra hepatic sources, mitochondrial damage and dilated cisternae of endoplasmic reticulum to be converted into lipid droplets after cerous chloride injection.
Kidney showed hypertrophy in epithelial cells, deformed Bowman's capsules, exfoliated nuclei in tubular lumen and leucocytic infiltration (Fig. 5). EGTA treatment showed well-formed glomeruli. The proximal tubules and collecting ducts had normal epithelial cells (Fig. 6). Day 21 showed maximum injury. Toxic effects of cerium caused intimate
association of podocytes with the capillary wall in Bowman's capsules (Fig. 7) and depletion in the number of cytoplasmic projections (Fig. 8). Steffee36
reported that the kidney under toxic effects shows hyline casts after rare earths exposure. Haematological and biochemical parameters indicate that cerium induced hepatotoxicity may be primarily responsible for severe effects on haemopoietic and renal tissues9
•13
• Various metabolic indices of cerium toxicity studied in the present investigation respond favourably to EGTA treatment. Cerium per se administration showed changes in blood parameters ' with maximum activity at day 14 followed by gradual recoupment by day 60. However, treatment with EGT A showed maximum changes in these parameters at days 7 followed by recoupment.
Thus, in the present study it was concluded that enzymes showed changes till day 21 when cerium exposed animals were administered EGT A. The toxic effects were restored considerably by day 7. This shows protective effect of EGT A against cerous sulphate toxicity. These results were substantiated by light and electron microscopic studies.
The efficacy of EGT A to mobilize cerium and restore the normal biochemical parameters may be attributed . to the available binding sites and the stability constant of the excretable metal chelator
Table J-Influence of EGTA on the activity of acid and alkaline phosphatase and succinic dehydrogenase of cerium exposed mice
[Values are mean ± SE from S observations in each group]
Groups Duration after treatment Acid phosphatase Alkaline phosphatase Succinic dehydrogenase (days) {mg P/l00gmlhr) (mgP/lOOgmlhr) (nmoles of K~e(CN)6)
Liver Kidney Liver Kidney Liver Kidney
Normal control 233.3±II.S 337.9O±17.5 84.7S1;4.39 2494.0Q±22S.00 47.2S±2.4S 30.02±1.9S Cerium 7 200.23±11.07 21S.94±11.40" 44.04±2.S3a 2283.33±193.2S 29.4S±1.61" 26.6S±1.70
P<O.OS "VS nonnal control. bvS cerium per se, '" significant.
00 00 o
-Z 0 ;; Z ...... tTl X ." IJ:l -0 .r en tTl
~ tTl ~ IJ:l tTl :;0 N
~
SHRIV ASTA V A & MATHUR: EFFECTIVENESS OF EGTA AGAINST CERIUM TOXICITY 881
Figs.I-4---{I) Effect of cerium on liver showing hypertrophy in hepatocytes (-.) X 400; (2) Effect of conjoint treatment on liver showing mitotic figurers (-. ) . in hepatocyte x 120; (3) Electronmicrograph of liver after cerium exposure showing dilatation of endoplasmic reticulum X 11500; and (4) Electronmicrograph of liver showing enlarged mitochondria in cerium treated mice xl 1500. [D=Dilatation of endoplasmic reticulum; ER=Endoplasmic reticulum; G=Glycogen; M=Mitochondria; N=Nucieus; NP=Nuciear pore]
882 INDIAN J EXP BIOL, SEPTEMBER 2004
Figs. 5-&-(5) After cerium exposure kidney showing deformed Bowman's capsule (~) x 120; (6) After conjoint treatment kidney showing well formed Bowman's capsule (~ ) x 120; (7) Electronmicrograph of kidney showing intimate relationship of a large podocyte with the capillary wall. Foot processes may be seen clearly attached on the capillary wall in cerium treated mice X 18000; and (8) Electronmicrograph of kidney showing hypertrophy of epithelial cell as a result of which the tubular lumen is reduced and depletion in tbe number of cytoplasmic projections in cerium treated mice X 23000. [CL=Capillary loop; FM-Filtration membrane; FP=Foot process; ICS=Inter cellular space; POD=-Podocytes; RBC=Red blood corpuscles; V=Vacuoles]
SHRlV ASTA V A & MATHUR: EFFECTIVENESS OF EGT A AGAINST CERIUM TOXICITY 883
complex37• Ahuja38 has determined the stability of
ternary complex formed between lanthanide and EGT A. Binding sites of cerium in EGT A are oxygen atoms and its co-ordination number is three. Ce EGT A complex is quite stable and does not dissociate as revealed by improved biochemical indices at longer durations. Takada et al. 39 reported excretion of cerium in both faeces and urine. Histopathological studies in the present study showed less necrotic changes in hepatic and renal tissues after EGT A treatment.
Acknowledgement One of the authors (SS) is thankful to U.G.c., New
Delhi for financial assistance.
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