Indian Journal of Experimental Biology Vol. 47, December 2009, pp. 955-963

Reversal of effects of intra peritoneally administered beryllium nitrate by tiron and CaNa3DTPA alone or in combination with α-tocopherol

Satendra Kumar Nirala1, Monika Bhadauria1*, Anil Kumar Upadhyay1, Ramesh Mathur1 & Asha Mathur2 1Reproductive Biology and Toxicology Laboratory, School of Studies in Zoology, Jiwaji University, Gwalior 474 011, India

2K R G (PG) College, Kampoo, Gwalior 474 009, India

Received 11 February 2009; revised 16 September 2009

To evaluate therapeutic efficacy of chelating agents tiron (Sodium-4,5-dihydroxy-1,3-benzene disulphonate) and CaNa3DTPA (Calcium trisodium diethylene triamine pentaacetic acid) in presence of α-tocopherol against beryllium induced toxicity, adult female albino rats were exposed to beryllium nitrate for 28 days followed by therapy with tiron (471 mg/kg, ip) and CaNa3DTPA (35 mg/kg, ip) alone and in combination with α-tocopherol (25 mg/kg, po). Results revealed non-significant fall in haemoglobin and total serum protein content while significant fall in blood sugar level and activity of serum alkaline phosphatase. On the other hand, significant rise in the activity of serum transaminases and LDH was noticed after beryllium administration. Significant increase in total and esterified cholesterol was found in liver and kidney after toxicity. Significant increase in lipid peroxidation and decreased level of reduced glutathione in both the organs showed oxidative stress due to beryllium exposure. Histopathological and ultrastructural observations of liver and kidney revealed lesions due to beryllium toxicity followed by recovery due to combined therapy. CaNa3DTPA showed moderate therapeutic efficacy; however, its effectiveness was enhanced with α-tocopherol to some extent. Tiron in combination with α-tocopherol exerted statistically more beneficial effects in reversal of beryllium induced biochemical, histopathological and ultrastructural alterations.

Keywords: α-tocopherol, Beryllium toxicity, CaNa3 DTPA, Combination therapy, Tiron

Beryllium induced lesions in experimental animals and industrial workers have been well documented. Beryllium is a naturally occurring element that is present in earth’s crust1. It was not known to have health hazards until it was used for industrial purposes. It is the lightest, bivalent and low-density hard metal having specific ability to add strength when small amount is added to copper and nickel to make alloys. Due to its unique chemical and physical properties, use of beryllium is increasing almost in every modern industry like aerospace, defense, electronics and ceramics. Because of its ubiquitous nature, it is found in coal, wood, foodstuffs and gemstones such as aquamarine and emerald2. The general population is exposed to naturally occurring beryllium from ambient air, drinking water, diet and smoking on a daily basis. Emissions from burning of fossil fuels i.e. coal and oil also increase beryllium level in atmosphere. Beryllium exposure can cause (i) acute pneumonitis⎯ a currently rare condition

caused by inhalation of beryllium salts or low fired beryllium oxide at concentration greater than 100 µg/m3; (ii) contact dermatitis from dermal contact with beryllium salts and (iii) chronic beryllium disease (CBD)- a potentially debilitating and fatal respiratory disease3. Ultimately beryllium gets accumulated in liver, kidney and bones and induces toxicity.

Therapeutic approach of chelating agents in combination with antioxidants for possible metal detoxification is an important aspect against metal poisoning. Tiron (sodium-4,5-dihydroxy-1,3-benzene disulphonate) is a relatively non toxic chelator, which has been tried in treatment of various metal poisonings, including uranium4, lead5, vanadium6 and beryllium7. CaNa3DTPA (calcium trisodium diethylene triamine pentaacetic acid) is a chelating agent belonging to polycarboxylate group of chelators, which is used to be applied in persons contaminated with plutonium or americium. Since CaNa3DTPA is partly absorbed following oral administration, it is usually given by injection8. α-Tocopherol is one of the most important lipophilic antioxidants, which acts as scavenger of free radicals

________________ *Correspondent author Telephones:+91-751-2442750 (Lab.); +91-9406970472 (Mobile) E-mail: bhadauria-monika @rediffmail.com

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within the membrane and protects unsaturated fatty acids from lipid peroxidation9,10. The present study has been designed to evaluate therapeutic efficacy of chelating agents tiron and CaNa3DTPA alone or in combination with α-tocopherol in amelioration of beryllium induced toxicity in rats. Materials and Methods

Chemicals⎯ Beryllium nitrate [Be(NO3)2] was purchased from Fluka (Switzerland), tiron, CaNa3DTPA and α-tocopherol acetate were obtained from HiMedia Laboratories Ltd. Mumbai, India. All the therapeutic agents were stored refrigerated in a desiccator to avoid oxidation and thermal decomposition. All other chemicals used in the study were of pure and analytical grade.

Maintenance of animals and their feeding⎯ Adult female albino rats of Sprague Dawely strain (8-10 weeks old having 130 ± 10 g body weight) were randomly selected from departmental animal facility where they were maintained under uniform husbandry conditions of light (14 hr) and dark (10 hr) at 25°±2° C and 60-70% RH. Animals were fed on standard commercially available pellets of animal diet (Pranav Agro Industries Ltd. New Delhi, India) and drinking water ad libitum. Experiments were performed in accordance with the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) Chennai, India and experimental protocols were approved by Institutional Ethics Committee (CPCSEA/501/01/A) of Jiwaji University.

Preparation of doses⎯ Beryllium nitrate was dissolved in triple distilled water making up doses of 1 mg/2 ml/kg. Doses of chelating agents CaNa3DTPA (35mg/2ml/kg) and tiron (471 mg/2 ml/kg) were prepared in 0.9% saline and pH was adjusted to 6.4 with sodium bicarbonate before administration. α-Tocopherol was dissolved in olive oil and doses of 25 mg/5ml/kg were administered orally with the help of intragastric rubber catheter. Selection of doses of toxicant, therapeutic agents and duration of treatment was based on earlier studies7,11,12.

Experimental design⎯ Rats (42) were divided into following 7 groups of 6 animals each:

Group 1: received sodium nitrate once a day daily for 28 days (1 mg/kg, ip) followed by saline (2 ml/kg, ip) for 5 days and served as normal control; Group 2: received beryllium nitrate [Be(NO3)2] once a day daily for 28 days (1 mg/kg, ip) followed by

saline (2 ml/kg, ip) for 5 days and served as experimental control; Group 3: received toxicant as in group 2 and treated with CaNa3DTPA (35 mg/kg, ip) for 5 consecutive days after toxicant administration; Group 4: received toxicant as in group 2 and treated with tiron (471 mg/kg, ip) for 5 consecutive days after toxicant administration; Group 5: received toxicant as in group 2 and treated with α-tocopherol (25 mg/kg, po) for 5 consecutive days after toxicant administration; Group 6: received toxicant as in group 2 and concomitantly treated with CaNa3DTPA (35 mg/kg, ip) and α-tocopherol (25 mg/kg, po) for 5 consecutive days after toxicant administration; Group 7: received toxicant as in group 2 and concomitantly treated with tiron (471 mg/kg, ip) and α-tocopherol (25 mg/kg, po) for 5 consecutive days after toxicant administration.

After 24 h of final administration, animals were euthanized under light ether anesthesia withdrawing blood in vials by puncturing retro-orbital venous sinus and finally serum was isolated. Liver and kidney were immediately excised, blotted free of adhering fluid and processed for biochemical studies, histopathological and ultrastructural preparations. Standard techniques were applied to assay following blood and tissue biochemical parameters.

Blood biochemical analysis Blood was immediately used for the estimation of hemoglobin13

and blood sugar14. Serum was used for the estimation of aspartate and alamine transaminases (AST and ALT)15, alkaline phosphatase (SALP)16, lactate dehydrogenase (LDH)17 and serum protein contents18. Serum albumin was estimated using E-Merck’s kit according to the manufacturer’s instructions.

Estimation of GSH and TBARS in liver and kidney Hepatorenal glutathione (GSH) measurement was performed using dithionitrobenzoic acid and optical density was recorded immediately at λ 412 nm19. The GSH level was calculated using an extinction coefficient of 13600/M/cm and expressed as μmol GSH/g tissue. The TBARS was assayed in liver and kidney for lipid peroxidation (LPO) and the LPO was expressed in terms of n mols TBARS/g tissue using an extinction coefficient of 1.56×105/M/cm20.

Hepatorenal lipid profile⎯ Method of Zlatkis et. al.,21 was followed for the estimation of total and esterified cholesterol in liver and kidney.

Histopathological observations: Optical microscopy⎯ For light microscopic observations,

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samples from the liver and kidney were fixed in Bouin’s fixative and processed routinely for embedding in paraffin. Tissue sections of 5 μm thickness were stained with hematoxylin and eosin (H & E) and examined under photomicroscope.

Ultrastructural observations: Transmission electron microscopy⎯ Small pieces of 1 mm3 of liver and kidney were fixed in Karnovsky’s fixative at 4°C for 18 h followed by washing with phosphate buffer. Post fixation was done with osmium tetraoxide (1%) followed by dehydration in acetone series. Subsequently, samples were embedded in epoxy resin and polymerized at 70°C for 20 h. Ultrathin sections were cut on Reichert Jung Ultracut-E Microtome. The sections were placed on uncoated grids and stained with uranyl acetate and lead citrate. Grids were examined under FEI Philips Morgagni 268D transmission electron microscope.

Statistical analysis⎯ Data were subjected to statistical analysis through one-way analysis of variance (ANOVA) followed by Student’s t-test22. Results were considered to be statistically significant at P≤ 0.05. % Protection was calculated by the following formula and the values are expressed as mean± SE:

Protection (%)= 1- D NT N−⎡ ⎤

⎢ ⎥−⎣ ⎦×100

where, D= drug, N= normal, T= toxicant Results

Blood biochemical analysis⎯ Blood biochemistry (Table 1) revealed significant fall in blood sugar level, serum albumin and activity of serum alkaline phosphatase, whereas significant rise was noticed in the activities of serum transaminases (AST and ALT) and LDH after beryllium administration; however, hemoglobin and total serum protein contents were declined non-significantly (P≤ 0.05). Tiron with and without α-tocopherol was found to be significant in maintaining blood sugar and serum albumin level towards normal (P≤ 0.05). Combination and single treatments significantly recovered altered activities of AST, ALT, LDH and SALP at 5% level while more than 80% protection was seen only with the combination of tiron and α-tocopherol.

Estimation of GSH and TBARS in liver and kidney⎯ Significant decrease in reduced glutathione and increase in lipid peroxidation in liver and kidney indicated beryllium induced oxidative stress (P≤ 0.05;

Table 1⎯ Effectiveness of CaNa3DTPA and tiron along with α-tocopherol against beryllium toxicity

[Values are mean ± SE of 6 rats in each group]

Groups Hb B. sugar S. protein S. albumin AST ALT LDH SALP

(g/100 ml) (mg/ 100 ml) (mg/100 ml) (g/ dl) (IU/L) (IU/L) (μ moles/min/L) (mg Pi/100 ml/h)

Control 15.2±0.84 108±5.97 37.2±2.05 5.63±0.31 68.3±3.77 43.2±2.38 42.2±2.33 206±11.3 Beryllium 13.2±0.72 71.3±3.94* 31.0±1.71 3.30±0.18* 120±6.63* 80.0±4.42* 137±7.57* 119±6.57*

Be+DTPA 13.8±0.76 80.2±4.43 33.4±1.84 3.60±0.19 100±5.52** 72.4±4.00 121±6.68 140±7.73 Protection (%) 30.0±3.86 24.2±4.18 38.7±3.38 12.8±4.81 38.6±3.38 20.6±4.38 16.8±4.59 24.1±4.19

Be+tiron 14.0±0.77 98.2±5.42** 35.0±1.93 4.10±0.22** 80.0±4.42** 54.4±3.00** 98.0±5.41** 167±9.23** Protection (%) 40.0±3.31 73.2±1.47 64.5±1.96 34.3±3.63 77.3±1.25 69.5±1.68 41.1±3.25 55.1±2.47

Be+α-toco 13.5±0.74 77.2±4.26 34.8±1.92 3.76±0.20 92.1±5.09** 60.4±3.33** 108±5.98** 172±9.50** Protection (%) 15.0±4.69 16.0±4.63 61.2±2.13 19.7±4.43 53.9±2.54 53.2±2.58 30.5±3.83 60.9±2.16

Be+DTPA+α-toco

14.0±0.77 82.7±4.57 35.4±1.95 3.90±0.21 91.3±5.04** 55.1±3.04** 106±5.85** 168±9.28**

Protection (%) 40.0±3.31 31.6±3.81 70.9±1.60 25.7±4.10 55.5±2.45 67.6±1.78 32.7±3.72 56.3±2.41

Be+tiron+α-toco

14.6±0.80 103±5.69** 36.0±1.99 4.60±0.25** 76.0±4.20** 48.2±2.66** 78.4±4.33** 197±10.8**

Protection (%) 70.0±1.65 86.3±0.75 80.6±1.06 55.7±2.44 85.1±0.82 86.4±0.77 61.8±2.11 89.6±0.57

F Variance 0.91 9.97# 1.30 13.5# 13.9# 18.4# 35.4# 12.5#

*Significant difference at P ≤ 0.05 compared with *control group; beryllium administered group. **#Significant F Variance at 5% level. Abbreviations: Hb (Haemoglobin); B. Sugar (Blood sugar); S. Protein (Serum protein); S. Albumin (Serum albumin); AST(Aspartate aminotransferase); ALT (Alanine aminotransferase); LDH (Lactate dehydrogenase); SALP (Serum alkaline phosphatase).

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Table 2). Combination of tiron and α-tocopherol was found to be most effective that reversed these variables towards normal significantly (P≤ 0.05) and showed more than 80% protection.

Hepatorenal lipid profile⎯ Significant raise was found in total and esterified cholesterol in liver and kidney (Table 2), which was lowered towards normal significantly by combined administration of tiron and α-tocopherol (P≤ 0.05).

Histopathological observations⎯ As compared to control (Fig. 1), liver of beryllium administered group showed high degree of vacuolation in hepatocytes and heavy leucocytic infiltration (Fig. 2). After co-administration of CaNa3DTPA and α-tocopherol, liver specimen persist with vacuolation, hyperchromatia of nuclei and central canal with debris (Fig. 3). Histology of liver after combined administration of tiron and α-tocopherol showed better cord arrangement, granulated cytoplasm, vasicular nuclei and normal Kupffer cells (Fig. 4).

Kidney of control group (Fig. 5) showing well formed glomeruli and uriniferous tubules with both basal and apical nuclei along with normal lumen. (Fig. 6) of beryllium administered group showed constriction of glomeruli, uriniferous tubules with hypertrophy and apical nuclei. Concomitant administration of CaNa3DTPA and α-tocopherol (Fig. 7) showed disruption of smooth epithelium, debris in the lumen of collecting duct. Apical nuclei

of uriniferous tubules were also observed. Combined administration of tiron and α-tocopherol (Fig. 8) showed more or less normal glomeruli and uriniferous tubules with basal and apical nuclei and wider lumen, and no leucocytic infiltration was observed.

Ultra structural observations⎯ Transmission Electron micrograph (Fig. 9) showed liver cell of control animal in which nucleus was smooth, rounded and vesicular with plenty of nuclear pores. In close association with nuclear membrane, there was rich network of endoplasmic reticulum and numerous mitochondria. There was rich distribution of glycogen bodies in the cytoplasm. Ribosomal endoplasmic reticulum was abundant. Beryllium administration for 28 days followed by vehicle for 5 days (Fig. 10) showed larger vacuoles in cytoplasm. The endoplasmic reticulum was lesser and shape of the nucleus became irregular. After co-administration of tiron and α-tocopherol (Fig. 11), hepatocyte had rounded nuclei surrounded by much mitochondria and endoplasmic reticulum. Nuclear membrane got regular in shape with prominent nucleolus.

Transmission electron micrograph of proximal convoluted tubule of kidney of control rat (Fig. 12) showed array of vertically oriented mitochondria and extensive infoldings of the basolateral plasmamembrane. Sub chronic exposure to beryllium caused severe damage in kidney (Fig. 13) showing less prominent and loosely arranged mitochondria,

Table 2⎯ Influence of CaNa3DTPA and tiron along with α-tocopherol against beryllium toxicity [Values are mean ± SE of 6 rats in each group]

Groups Lipid peroxidation Glutathione Total cholesterol Esterified cholesterol (n moles TBARS/mg protein) (µ moles/g tissue) (mg/100mg ) (mg/ 100mg)

Hepatic Renal Hepatic Renal Hepatic Renal Hepatic Renal

Be+DTPA 0.448±0.02** 0.812±0.04 6.83±0.37 3.42±0.18 0.200±0.01** 0.198±0.010 0.157±0.008 0.101±0.005 Protection (%) 58.1±2.31 17.5±4.55 21.0±4.36 9.09±5.02 43.6±3.11 28.0±3.98 5.49±5.22 4.87±4.25

Be+tiron 0.413±0.02** 0.588±0.03** 7.24±0.40 4.28±0.23** 0.178±0.009** 0.189±0.010 0.115±0.006**0.065±0.003**Protection (%) 67.0±1.81 48.2±2.86 41.1±3.25 64.9±1.93 63.6±2.01 37.0±3.48 51.6±2.67 48.7±2.83

Be+α-toco 0.409±0.02** 0.716±0.03** 6.95±0.39 3.50±0.19 0.190±0.010** 0.212±0.011 0.110±0.006** 0.098±0.004 Protection (%) 68.1±1.76 30.6±3.83 26.9±4.03 14.2±4.73 52.7±2.61 14.0±4.75 57.1±2.36 8.53±5.05

Be+DTPA+α-toco

0.332±0.01** 0.782±0.04** 7.13±0.38 3.68±0.20 0.168±0.009** 0.176±0.009** 0.105±0.005** 0.089±0.004

Protection (%) 87.7±0.67 21.6±4.33 35.7±3.54 25.9±4.09 72.7±1.50 15.0±2.76 62.6±2.06 19.5±4.44

Be+tiron+α-toco

0.312±0.01** 0.392±0.02** 8.23±0.45** 4.72±0.26** 0.154±0.008** 0.142±0.007** 0.079±0.003**0.044±0.002**

Protection (%) 92.8±0.39 75.0±1.37 89.7±0.56 93.5±0.35 85.4±0.80 84.0±0.88 91.2±0.48 74.3±1.41

F Variance 37.0# 56.2# 4.05# 10.0# 14.6# 15.2# 34.0# 61.1#

*Significant difference at P ≤ 0.05 compared with control group; **Beryllium administered group. #Significant F Variance at 5% level.

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deformed nucleus, less infoldings of basolateral plasmamembrane and degeneration of the basement membrane. Co-treatment with tiron and α-tocopherol (Fig. 14) showed prominent and vertically arranged mitochondria, well-formed nucleus and deeper infoldings of basolateral plasmamembrane into the cytoplasm. Discussion

Present investigation had been carried out to compare efficacy of chelating agents individually and in combination with α-tocopherol. Results revealed severe alterations in histopathological, blood and tissue biochemical variables after intraperitoneal administration of beryllium. Reduced synthesis of heme and globulin proteins due to beryllium poisoning resulted in a consequent decrease in hemoglobin content of erythrocytes23,7. Significant fall in blood sugar level after beryllium intoxication

may be due to liver damage and increase in blood lactic acid, which resulted in hypoglycemia. Significant raise in the serum transaminases and LDH may not only be due to phagocytosis and necrosis in tissues of liver and kidney24,25 but also due to alterations in membrane permeability, which permitted rapid out flow of these enzymes26,27. Increased level of AST probably elevated the oxidation of aspartic acid that gets transformed into cholesterol and thus, increased level of cholesterol was noticed in liver and kidney. Inhibition in the activity of serum alkaline phosphatase during beryllium toxicity may be due to the displacement of magnesium ion (Mg2+) by beryllium ions (Be2+)28. In vivo and in vitro studies have suggested that Be2+ ions always compete with Mg2+ ions 29, which results in the lowering of activity of alkaline phosphatase30. Inhibition may also be due to the formation of insoluble phosphate which further interferes with the

Figs. 1-4 ⎯ Liver of (1)-normal control; (2)-beryllium treated group; (3)-Be + DTPA + α-tocopherol treated group; and (4)-Be + tiron + α-tocopherol treated group.

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absorption of phosphate in gastrointestinal tract. Administration of chelating agents may reduce beryllium ion concentration in liver and kidney, whereas concomitant administration of α-tocopherol inhibited oxidative phenomenon during toxicity, which helped in reversing aforesaid parameters towards normal.

Bulk of the circulating beryllium binds with plasma globulin because of its apparent protein binding property and forms stable beryllium protein complex. In this way, a large amount of beryllium gets transported to various organs and causes damage27,23. Thus, impaired functioning of liver declined protein synthesis and so its level in tissues. Hypoalbuminemia may result from increased catabolism31 during beryllium toxicity. Lipid peroxidation is regarded as

one of the basic mechanism of tissue damage caused by free radicals32. In the present study, enhanced level of TBARS in liver and kidney was strongly reduced by co-treatment of tiron and α-tocopherol rather than CaNa3DTPA or its combination. Since, tiron is SOD mimetic, it may have dual effects; one is quenching the adverse manifestation of beryllium ions through chelation33 and other is scavenging superoxide anions34, whereas α-tocopherol is able to quench the LPO chain and protect membrane from attack of free radicals. The GSH, a non-protein thiol is involved in many cellular processes, including detoxification of endogenous and exogenous compound35. In the present studies decreased glutathione level after beryllium administration may be due to its increased

Figs. 5-8⎯ Kidney of (5)-normal control; (6)-beryllium treated group; (7)-Be + DTPA + α-tocopherol group; and (H&E 200×) (8)- Be + tiron + α-tocopherol group (H&E 200×). [V=Vacuolation, LI=Lymphocytic infiltration, H=Hyperchromatia of nuclei, GN=Glomeruli normal, CG=Constriction in glomeruli, AN= Apical nuclei, H=Hypertrophy, D=Disruption of smooth epithelial cell, DB=Debris, BN=Basal nuclei, AP=Apical nuclei (H & E 200×)]

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utilization by hepatocytes36,37 because GSH acts as a scavenger for toxic chemical agents38 besides as natural antioxidant. Combined administration of chelating agents and α-tocopherol counteracted free radical mediated cell injury during severe hepatorenal

damage conditions and have elevated GSH level, which in turn helped in mitigating tissue damage. The histopathological and ultrastructural observations basically supported the results obtained from serum and tissue biochemical estimations.

Figs 9-14⎯ Electron micrograph of (9)-control liver (3500×); (10)-beryllium intoxicated liver (5600×); (11)-liver after combined treatment of tiron and α-tocopherol (1100×); (12)-Electron micrograph of control kidney (1400×); (13)-beryllium intoxicated kidney (3500×); and (14)-kidney after combined treatment of tiron and α-tocopherol (1400×). [N=Nucleus, ER=Endoplasmic reticulum, NP=Nuclear pore, G=Glycogen, V=Vacuoles, Lg=Lipid globule, Nu=Nucleolus, M=Mitochondria, I=Infolodings of basolateral membrane, BM=Basement membrane].

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It can be hypothesized that chelators may reduce beryllium body burden and simultaneously exogenous antioxidant α-tocopherol reduced oxidative stress leading to fast recovery in damaged tissues. In the present study CaNa3DTPA showed moderate therapeutic potential, whereas α-tocopherol enhanced its effectiveness to some extent. Tiron in combination with α-tocopherol exerted more beneficial effects over a combination of CaNa3DTPA and α-tocopherol treatment against beryllium induced biochemical, histopathological and ultrastructural alterations suggesting that combination therapy of tiron and α-tocopherol could be preferred as a better choice in treatment of beryllium induced toxicity. However, chelator especially tiron should be further investigated at relatively lower doses as potent therapeutic agent against beryllium intoxication. Acknowledgement

One of the authors (SKN) is thankful to Indian Council of Medical Research, New Delhi for financial assistance through Research Associateship and Jiwaji University for providing laboratory facilities. References

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