Indian Journal of Experimental Biology Vol. 42, August 2004, pp. 798-802

Analysis of time-dependent recovery from beryllium toxicity following chelation therapy and antioxidant supplementation

Sonia lohri, Sadhana Shrivastava, Pragya Sharma & Sangeeta Shukla*

Laboratory of Toxicology & Reproductive Biology, School of Studies in Zoology, Jiwaji University, Gwalior, 474 011, India

Received 28 April 2003; revised 20 April 2004

Efforts have been made to minimize the toxic effect caused by beryllium. Adult cyclic rats of Sprague Dawley strain were administered a bolus dose of 50mglkg beryllium nitrate intramuscularly. The chelation therapy with glutathione (GSH), dimercapto propane sulfonic acid (DMPS)+ selenium (Se) and D-Penicillarnine (DPA) + Se was given for 3 days followed by a rest of 1,3 and 7 days respectively. The results revealed a significant faU in the blood sugar level, serum alkaline phosphatase activity, serum proteins. A significant rise in the transaminases i.e. aspartate aminotranferase and alanine aminotranferase pattern is indicative of leakage of enzymes from liver resulting in alterations in the cell permeability. A rise in the hepatic lipid peroxidation activity is a direct indication of oxidative damage resulting in free radical generation. Results of the distribution studies by atomic absorption spectrophotometry reveal an increased concentration of beryllium in liver and kidney followed by lung and uterus. The relative ability of 3 chelating agents to act as antagonists for acute beryllium poisoning have been examined in liver, kidney, lungs and uterus. The appreciable change in the beryllium concentration in various organs is duration-dependent during the entire period being highly significant after 7 days rest. From the biochemical assays, and distribution studies it can be assumed that DPA+Se was the most effective therapeutic agent followed by DMPS+Se and GSH. Thus it can be concluded that DPA+Se is a better therapeutic agent as compared to DMPS+Se and GSH.

Keywords: Antioxidant supplementation, Beryllium toxicity, Chelation therapy, Time-dependent recovery.

Beryllium, a divalent metal ion is toxic to both man and animals and has potential to produce disease wherever incorrect practices prevail 1.2 • The potential hazards of exposure to beryllium compounds were first recognized in 19303

. Occupational exposure to beryllium may lead to symptoms of berylliosis, dermatitis, granulomatosis, pneumomtIs, cardiac failure, hepatomegaly, splenomegaly, cyanosis, finger clubbing and disturbance in immune system. This metal is known to act as an antigen after complexing with the host proteins and also act as an adjuvant to the immune response against other antigens4

. Workers exposed to beryllium work areas though sanitized to meet OSHA standards have reported a statistical rise in blood beryllium antibody titres5

•6

. Chronic beryllium disease, which results from occupational exposure to particulate beryllium, is characterized by the development of lung granulomas and progressive pulmonary fibrosis 7.

In the present study, an attempt has been made to reduce toxicity caused by beryllium through chelating

*Correspondent author Phone: 91751-5016750 Fax: 91-751 -2341450 Email-dcsshukla@hotmail.com

agents and mmlIllize oxidative stress by the use of aSH and Se. Research into the possibility that selenium may work as a general detoxifying agent is relatively new. Selenium, an antioxidant prevents the oxidative degradation of biological molecules. It is believed to be an essential element in mans. One well­defined function of Se is as an important element in glutathione peroxidase. This enzyme plays a significant role in protecting tissues from peroxidative cell damage by catalyzing peroxides9

-11

.

Materials and Methods Animals and Chemicals-The chelating agents viz.

glutathione (aSH), 2,3 dimercapto propane sulfonic acid (DMPS), D-Penicillamine (DPA) and sodium selenite were procured from Sigma Chemicals Co. (St. Louis, MO) and beryllium nitrate was procured from Fluka, Switzerland. Adult female albino rats of the Sprague Dawley strain (150±1O g) were selected from the animal facility of the department. Animals were randomized to treatment groups and kept under constant conditions of humidity, photocycle (14:10 hr L:D schedule) and temperature (24°±2°C). They were fed standard pellet diet (Lipton India Ltd.), metal content of the diet in ppm dry weight was Cu 10, Fe

JOHR! et al.:THERAPEUTIC APPROACH AGAINST BERYLLIUM INTOXICATION 799

70, Zn 45 and Co 5 and drinking water ad Libitum. Both chelating agents were prepared fresh and the pH was adjusted to 6.4 with sodium bi-carbonate. The animals (25) were divided in five groups of 5 animals each.

ExperimentaL design-The adult cyclic rats were administered a bolus dose of 50 mg/kg body weight beryllium as beryllium nitrate dissolved in distilled water. Five animals received normal saline and served as control (Group 1).

Group 2 comprised animals that were administered beryllium nitrate solution at a dose of 50 mg/kg (im). The other groups (Groups 2-5) also received beryllium nitrate and in addition were treated with chelating agents viz glutathione (ip) (group 3), OMPS (ip) (group 4), OPA (orally) (group 5) in combination with sodium selenite (sc) for three days. Five animals from each group were sacrificed after 1, 3 and 7 days rest. The treatment details in various groups are as follows:

Group 1: 4ml/kg normal saline (Control) Group 2: Beryllium per se (50mg/kg body weight) Group 3: Be+ GSH (0.3mM/kg body weight) Group 4: Be+OMPS (0.3mM/kg body weight)+

Selenium (0.5mglkg body weight) Group 5: Be+OPA (0.3mM/kg body weight) +

Selenium (0.5mg/kg body weight)

Immediately after necropsy, blood was collected by puncturing the retro-orbital sinus. Biochemical estimations were done spectrophotometrically (Systronics UV visible Spectrophotometer 108). Blood sugar was evaluated by the amount of cuprous formed by reacting with phosphomolybdic acid using the method of Asatoor and King l2. Serum trarisaminases were estimated by the method of Reitman and Frankel 13, serum alkaline phosphatase was estimated by reading the absorbance at 620nm by the method of Fiske and Subbarowl4, and protein content in serum was determined by the method of Lowry et aLls. Hepatic lipid peroxidation was estimated l6 by the reaction of MOA, a degradation product of peroxidized lipids with TBA to produce TBA malondialdehyde chromophores, which has been an index of lipid peroxidation.

MetaL estimation-The tissues i.e. liver, kidney, lungs and uterus, were digested by the method of Bokowski17

• The distribution studies were performed by atomic absorption spectrophotometry where the concentration of beryllium in each organ was estimated.

StatisticaL anaLysis-The data were analyzed by Student's t test followed by two-way analysis of variance lS, in which comparison was made between various treatments as well as various durations.

Results and Discussion In the present investigation, beryllium intoxicated

rats were given treatments of various chelating agents in combination with an antioxidant selenium for 3 days followed by a rest of 1,3 and 7 days respectively. The results reveal a duration dependent response. The haemoglobin % showed a decline at all the three durations but it was not significant, however, Se in combination with OPA proved to be effective when compared with the other two chelators. The alteration in the haemoglobin % profile after the administration of beryllium nitrate is potentiated by Shukla et aL. where a fall in Hb% was observed on beryllium exposurel9

• The blood sugar level showed a tremendous fall after beryllium exposure, which is very significant after 7 days rest. Administration of GSH, OMPS and OPA+Se prevented hypoglycaemia to a considerable extent. A noticeable recovery was observed with DPA+Se in both the parameters after 7 days rest (Table 1). A significant fall in the blood sugar level after toxicant administration may be due to the dysfunctional and dystrophic changes in the liver and increased production of blood lactic acid may result in hypoglycaemia. These findings were supported by Mathur et al.2o Beryllium induced inhibition of serum alkaline phosphatase and serum proteins at all the three durations showed maximum fall after 7 days regimen. With OPA + Se the recoupment was quite significant and the enzymatic activity was towards control (Table 1). Inhibition in the activity of alkaline phosphatase during beryllium toxicity is attributed to displacement of Mg2+ by Be2+. It is held that Co, Ni and Mg antagonized the inhibitory effect of beryllium on serum alkaline phosphatase activity21-24.

Transaminases, which are the reliable marker enzymes of the liver function, showed duration dependent response depicting peak value after 7 days of treatment. GSH followed by OPA+Se prevented · leakage of these enzymes thereby showing anti oxidative efficacy of these agents (Table 1). The therapeutic agents may protect against acute organ dysfunction and cellular injury thereby preventing the leakage of enzymes. The alteration in the permeability of the cell membrane and the inhibition of the ion transporting system results in the disturbances in the

800 INDIAN J EXP BIOL, AUGUST 2004

fluid movement. Vacher et al. reported increase in the AST, ALT and ~ glucouronidase activity after beryllium exposure22

• It may be due to phagocytosis or necrosis of the liver. Various authors observed a remarkable elevation in the transaminases pattem20

,25.

Thus, the antioxidants administered proved their efficacy in inhibiting their efflux in the blood.

Toxicant induced stress at a biochemical level is based on the production of free radical. Oxidative stress caused by toxicants in biological systems may be involved in a variety of disease states and toxic reactions, thereby contributing indirectly to injury. The results reveal a tremendous rise in lipid peroxidation process after beryllium administration

Table I-Effect of im administration of beryllium nitrate (50 mg/kg once only) followed by chelating agents for three consecutive days on the blood biochemistry of adult female rats

[Values are as mean ± SE of 5 rats in each group]

Treatment Days Hb% Blood Sugar g % mgllOOml

A 14.1±1.3 91.8±8.4 B 13.3±1.2 71.4±4.6 C 13.9±1.2 72.4±5.5 D I 4.5± 1.6 71.6±5.3 E 14.9±1.6 78.6±4.6

A 3 14.7±1.3 95.8±4.6 B 13.7±1.2 61.8±3.0 C 14.2±1.l 71±5.6 D 13.9±1.9 66.6±3.2 E 14.2±O.8 64.2±3.8

A 7 14.9±1.2 9O±10.3 B 1O.8±O.5' 6O.4±4.0' C 11.6±1.l 73.4±2.3H

D 14.2±1.3H 72.2±1.9 E 13.8±1.2" 81.6±3.8"

F Variance Ratio Days 3.3"1 16.3-

Treatments 11.2- 2.34"1

Treatment Days AST ALT lUlL lUlL

A 64.5+ 5.5 47.7±2.7 B 87.8± 3.9' 51.4±2.7 C 69.0±_4.8" 45.6±1.9 D 75.0±3.2H 50.6±3.2 E 74.6±2.9" 50.0±3.3

A 3 67.2±5.2 49.8±3.8 B 88.2±3.9' 60.8±4.3 C 69.4±3.8" 52.0±3.5 D 69.0±2.2H 53.2±1.5 E 56.0±3.2 54.3±3.6

A 7 64.2±4.4 48.4±1.5 B 71.0±2.9' 74.6±3.8 C 49.8±2.5 59.6±4.3 D 70.6±2.1 68.8±4.2 E 48.6±3.5" 63.0±1.8

F Variance Ratio Days 29.9* 18.9*

Treatments 5.48* 2.9* A=Control; B=Be; C=Be + GSH; D=Be + DMPS + Se; E=Be + DPA + Se.

Serum Proteins mgl I OOml

41.6±3.5 37.6±2.1 38.2±2.5 35.4±1.l 38.0±2.7

40.3±1.9 34.0±1.8' 35.0±1.9 35.0±1.l 36.4±1.3

42.8±2.3 36.4±1.0' 37.0±1.5 36.0±1.l 38.2±1.8

2.03"' 5.47"'

Serum ALP mg Pill00mIlh

206.8±9.3 195±10.7 212.4±4.7

170.2±5.6" 200.2±7.4

212.4±1O.1 I 77.0±9.30 179.2±11.7 185.6±12.8 192.2±1O.1

209.1±13.1 149.2±1.80" 158.0±5.20 167.6±4.40 189.6±12.8

2.03"' 5.47*

#P ~ 0.05 when compared with control group. ## P ~ 0.05 when compared with Be treated group. * = Significant F value at 5% level. ns = Not significant F value at 5% level.

JOHRI et ai.:THERAPEUTIC APPROACH AGAINST BERYLLIUM INTOXICATION 801

(Table 2), which increased linearly reaching its peak at 7 days regimen. These changes were restored considerably with chelation therapy. The combination of a chelator DPA with antioxidant Se proved to be very efficacious in restoring the levels towards normal. aSH and DMPS+Se were also effective in

Table 2-Effect of i.m. administration of beryllium nitrate (50 mg/kg once only) followed by chelating agents for three consecutive days on the blood biochemistry of adult female rats. (Values are mean ± SE of S rats in each group.)

Treatment

A B C D E

A B C D E

A B C D E

F Variance ratio

Days

3

7

Days 32.4" Treatments 2.07"S

A, B, C, D-Same as in Table 1

Hepatic LPO (n moles of MDAlmg

protein)

0.29±O.02 0.49±0.02# 0.39±O.OS 0.40±0.06

0.3S±0.02##

0.29±O.02 0.4O±Q.03# 0.4S±O.03 0.39±O.03

0.33±O.02##

0.30±0.02 0.4S±0.03# 0.3S±O.04

0.36±O.04## 0.33±O.02##

# P ~ O.OS when compared with control group. ## P ~ O.OS when compared with Be treated group. * = Significant F value at S% level. ns = Not significant F value at S% level.

recouping this variable thereby reducing oxidative stress. A significant rise in the lipid peroxidation as seen in the present investigation is also supported by various authors 19,25. Increased level of lipid peroxidation is a direct indication of the microsomal lipids in the liver. It may also be correlated with the reduction in the antioxidative defense enzyme systems. As free radicals are damaging in nature, use of antioxidants was thought to be an alternative to combat oxidative stress. Selenium, an antioxidant prevents the oxidative degradation of biological molecules thereby conferring its protective effect.

Table 3 presents the effect of chelators on the tissue distribution of beryllium. The beryllium retention in tissue varied among the organs. It was observed that the concentration of beryllium was highly elevated in liver and kidney followed by lung and uterus. All the chelators were effective in removing beryllium from the tissues when compared to beryllium per se group, however, values indicate that DPA+Se was significantly effective when compared with other chelators. Flora et al.25 stated that the rate of removal of the metal ion after DMPS treatment was greater than DMSA in beryllium poisoning. They also reported significant recoveries in the biochemical variables following administration of DMPS, which indicated that the beneficial effect of chelation of toxic metal ion by thiols is unquestionable.

The efficacy of each of these therapeutic agents may be attributed to available binding sites and the stability constant of the excretable metal chelator complex. aSH plays a major role in the detoxification strategy, serving as a nuc1eophile thereby forming drug conjugates. Some cells have a Na+ dependent uptake system for aSH and that this system allows cells to use exogenous aSH to supplement

Table 3-Effect of Lm. administration of beryllium nitrate (SO mg/kg once only) followed by chelating agents for three consecutive days on the distribution studies (ltg/g)

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

Liver Kidne:t Lungs Uterus 1 day 7 day 1 day 7 day 1 day 7 day 1 day 7 day

Control O.SO±O.06 O.Sl±O.04 O.70±0.09 O.72±O.OS 0.27±0.04 0.31±0.OS 0.10±0.02 0.12±0.03

Beryllium 13.0±1.0# 22.3±2.1# 1O.4±1.2# IS.3±1.9# 1.5±1.3# 15.9±1.6# S.6±O.6# 1O.S±1.6#

Be+GSH 7.8±0.S## 8.8±0.1## 4.9±O.OS## S.S±O.6## S.6±0.S## 6.3±0.7S## 5.0±0.46. 7±0.5##

Be+DMPS+Se 12.0 ±l.S 4.9±O.S## 6.2±0.6## 3.3±0.3## 4.9±0.S## S.8±O.S## 4.3±O.54. S±O.S##

Be+DPA+Se 6.5±1.2## S.O±O.S## 3.3±0.3## 4.I±O.S## S.l±O.S## 6.I±O.7## 3.9±O.3## 4.3±0.4##

# P < O.OS when compared with control group. ## P < O.OS when compared with Be treated group.

802 INDIAN J EXP BIOL, AUGUST 2004

endogenous synthesis26• Orally supplied GSH is not a

major determinant of blood plasma GSH concentration27

.28

• However, regular supply of GSH potentiates cell survival, helping in maintaining the cell integrity, thus decreasing the susceptibility of the membrane to further exposure. Thus, GSH protects against beryllium toxicity either by direct conjugation with the metal ion or inhibition of free radical generation. The intracellular chelation by GSH eliminates the beryllium ion, which might be the first mechanism of protection offered by it along with its antioxidative efficacy. The limitations of GSH is possibly due to its large structure which might result in the folding of the complex (Be:GSH) formed.

The viability of Be:DPA complex is more due to the fact that 80% of DPA can be eliminated in the urine within 24 hrs. The complex is more stable as it forms five membered rings alternatively. Acute LD50 of DPA in rats is higher, thus establishing its possibility as a better chelate. Selenium prevented oxidati ve degradation of biological membranes by catalyzing the donation of hydrogen atom from reduced glutathione to reactive and potentially damaging peroxide radical hence conferring its protective action.

Thus, it is concluded that there is a hepatic injury to the tissue on administration of beryllium that is reversed considerably with therapeutic agents. DPA+Se, GSH and DMPS+Se are similar in their ability to reverse the toxicity of Be with regard to hepatic LPO. The action of DPA+Se appears to be faster in some aspects.

Acknowledgement The authors feel indebted to Prof. R. Mathur,

School of Studies in Zoology, Jiwaji University, Gwalior, for suggestions and support. The financial assistance from MPCST, Bhopal and CSIR, Delhi is acknowledged.

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