Indian Journal of Expcrimcnla l Biology Vol. 40, May 2002, pp. 564-570

Modulation of acute cadmium toxicity by Emblica officinalis fruit in rat*

S Khandelwal , LJ ShuklJ & R Shanker

Industrial Toxicology Resea rch Centre Luck now 22600 I, India

E-mail : root@itrc.crnet.in ; Fax No. 0522-228227

Received 30 Norelllber 2000: revised 13 NOI'e lllber 2001

The efli cacy of Elllblic(I (i(jrcil/ (l lis in modifying the acute cy totox ic ity of cadmium in ma lc ra ts was evaluated. Oral admini stl'ati on of Elllblic(I fruit juice (500 mg/kg, b.w.) for 8 days fo ll owed by a single tox ic dose of Cd as CdCl 2 (3 mg/kg, b.w. ip), considerabl y reduced the mortality in rats as well as prevented to some extent the cadmium induced hi stopa­thological damage in testi s, liver and kidneys . Biochemical in vestigation also revea led reduced levels of Cd induced serum glutamate oxal oacetate transam inase, glutamate pyruvate transaminase and gam ma glutamy ltranspept idase. The enhanced level s of Cd and lipid peroxidation in li ver, kidney , and testes andmetallothionein and total su lphyd ryl in liver and kidney by Cd were signifi cant ly reduced by Elllblica pretreatment. These results suggest eytoprotective potential of Elllblica fruit in acute cadmium toxici ty wh ich cou ld be due to its multiple rol e in biological system.

Cadm ium (Cd) highl y toxic to both human and ani ­mal s, is ubiquitous due to its wide application in elec­troplating and galvani zing, as a colour pigment in paints, batteries, plastic and fertili zer industries, ciga­rette smoke, air pollution and most foods 1.2. It has been class ifi ed as human carcinogen by International Agency for Research on Cancer3. Genera ll y tes ti s, li ver and heart are most damaged fo llowing acute ex ­posure to Cd~ ·5 whereas renal toxicity predominates f h . I I I . . 67 a ter c ronic ow eve pOi soning' .

Although, hi gh incidence of low level ex posure to cadmi um takes place, proper therapeutic in tervention remains obscure. Most of the syn thet ic antidotes in the management of Cd tox ici ty have met with limited success due to their inherent tox icity, non spec ificity causing essenti al meta l depletion and low accept ibil­ity by exposed subjcctss.

Free rad ical scavengers and anti ox idants are useful in protecting Cd tox icit/ . Fari ss tO has studied the cy­toprotec ti ve properly of a-tocopheryl succinate in Cd tox icity. Ascorb ic ac id, too ac ts as a pro-oxidant or an oxygen rad ical scavenger depending on an optimal threshold dose for effective antagoni sm by thc chemicai li

.

There are many reports of correlati on between hi gh consumption of fruit and vegetabl es or of spec ific di e­tary anti ox idants (v itam in C, carotenoids, vitamin E) and a re lat ively low incidence of severa l cancers 12 . Dietary antiox idanLs, in general, act- by re-

*ITRC Publication No. 2099

moving reactive oxygen spec ies before they have a chance to cause damage to biological molecules.

Fruits of ElIIblica offi cinalis Gaertn . (Fam il y Eu­phorbiacea) co mmon ly known as 'am i a' or the Indi an gooseberry has been ex tensively used in Indian Ay­urved ic and Sidha system of trad itional medicine for the treatment of wide spectrum of diseases . It is the constituent of several marketed herbal preparat ions such as Chyavanaprasa, Brahma Rasayana, Amalak Hari tak i, Triphala, Septi lin etc. Some of the properties of the fruit ex perimenta ll y proved include anti fungal , anti bacteri al antidi abetic, antipyret ic, anti ox idat i ve, anticlas togeni c, hepatoprotecti ve etc. 13.IS.

ElII blica fruit contains tannins such as gallic ac id, ell ag ic acid, albumin , crude ce llulose, nicotinic acid , am ino ac ids like glutamic acid, proline, aspart ic ac id, alanine and lys ine, minerals, Cr, Zn, Fe and Cu apart from the· hi ghest amount of vitamin C in natural form . Some cytoki ne- li ke substances also identified are zeatin , z. riboside, z. nucleotide.

Desp ite its ex tensive med icinal use, li mited knowl­edge is available regarding its role in heavy metal tox­icity. Few studies have been carri ed out on its modify­ing effect aga inst nickel, lead and aluminium clasto­genicity in miceI 3 .1 ~ . However, its antioxidati ve and cytoprotect ive potential against Cd tox icity remains ulle )( plorecl . Therefore, the purpose of thi s study was to delineate its role in ac ute Cd-induced hepat ic, renal and tes ti cular damage in rat.

KHA NDELWAL el al.: CA DMI UM TOXICITY & EMBLICA OFFICINAU S 565

Materials and Methods

Cheillicals Cadmium chloride was obtained from Sigma (St.

Loui s, MO, US A) . Fresh El1Iblica fruits procured from Nati onal Botanical Research Institute, Lucknow, were grated, weighed and crushed in pestle and mo­tor. The juice was pressed through cheese cloth and a 50% solution was prepared in double di st ill ed water. Other chemi cals were of analyti cal grade.

Animals and frealillenf Forty eight healthy male albino rats of Druckrey

strain of ITRC colony were used. For the first sel of experiment , 24 animals were di vided into four groups of six rats each. ElIlblica juice (500 mg/kg/5 ml ) was fed orall y to two groups of 6 rats each , daily for 8 days. On the las t day, one hour after the El71blica dose, Cd as CdCI 2 (3 mg Cd/kg/ml in normal saline) was injected i.p to one group and the other group of El71blica was inj ected saline. One set of 6 rats were admini stered Cd and the remaining 6 animals were injected saline and served as co ntrol s. The mortality pattern was observed over a peri od of 48 hrs. (The LDso of Cd is 3.55 mg/kg).

Another set of 24 rats were treated exactly as above and sacrifi ced after 6 hours of Cd treatment. Blood fro m jugul ar ve in was coll ected for serum separat ion and the ti ssues (li ver, kidney and testi s) were di s­sected and cleaned free of ex traneous materi al. A part of li ver, half kidney and one testes were immedi ately put in 10% neutral buffe red formalin for hi stopa­tholog ica l exa minati on. A portion of liver, half kidney and a piece of testes were kept at -20°C for Cd es ti­mati on and a porti on of all the th ree ti ss ues was used for the assay of lipid perox idati on and total sulphydryl content.

Assay oj lipid peroxidation, 10lal sulpllydryl (SH), serulll enZYlIles, lIl etal/othioll ein (MT) and cadllliulIl Ie lie Is

A 20% homogenate of li ver, kidney and testes was prepared in 1.1 5% KCI and a 10% homogenate in 0.02M EDTA under ice-cold conditions for lipid per­ox idati on and total sulph ydryl level, respec ti ve ly , and estimated the ve ry same day. A part of the homoge­nate in KCI was spun at 100,OOOg (60 min ., O°C) and the supernatant frac ti ons used for MT determinat ion. Serum was refri gerated and used for biochemi cal pa­rameters the next day.

The esti mati on of lipid perox idati on was monitored by malondi aldehyde (MDA) formation by the method of Ohkawa el al.19 and total SH by Sedlek and Lind­say20 . The clini ca l biochemi ca l analys is in seru m was performed on Autolab Autoanalyser of Boehri nger Mannheim, Germany. An alys is was carri ed out at 37°C in serum samples according to the instructi ons of the manufac turer (Boehringer Mann heim, Appli ca­ti on Sheets, 1995). Briefly, glutamate oxaloacetate transaminase (GOT)and glutamate pyru va te transami­nase (GPT) ac tivity were determined according to the Intern ational Federati on of Cl inical Chemistry Kineti c method , alka line phosphatase (A LP) using AMP buffer and gam ma glutamy l transpcptidase (GGT ) activity according to the kinetic method of Szasz.

Metallothi onein (MT) in li ver, kidney and testes was est imated usi ng Cd-Chelex assay21 with some modifi cations. Non radi oacti ve Cd as CdCl2 ( I mM) was used for saturation of MT in the assay and the Cd content in the supernatant was measured on Varian Spectr AA. 250 Plus Atomic Absorpti on Spectropho­tometer. Wet ac id di gestion proc dure for measuring Cd levels in li ve r, kidney and tes tes using HNO, and HCl04 was carri ed out and the resultant so lution was made up with deioni zed water. Cadmium was ana­lysed on Vari an AAS at 228.8 nm wave length.

Histological allalysis oj lissue daJllage For hi stopatholog ica l assessment of hepati c, renal

or testicul ar damage, ti ssues we re fixed in 10% neu­tral buffered formalin , embedded in paraffin , sec­ti oned and stained with hematoxy lin and eos in22

.

Slatistical allalysis Signi ficance of mean values of different parameters

between th0 treatmen t groups were analysed using One Way analys is of vari ance, after ascertaining the homogeneity of va riance betwee the treatments. Pair wi se compari sons were done by ca lculating the least signifi ca nt difference .

Results A single intraperitonea l injection of 3 mg Cd/kg

caused 66% mortality over a period of 48 hrs. How­ever, Cd ind uced mortality was marked ly reduced when the r:ilS were pretreated with Emblica fr ui t juice (500 mg/kg, oral) for 8 days (Tab le I).

On hi stopathological examination , Cd alone caused typi ca l spectrum of tes ti cul ar les ions in all the animals whi ch compri sed of edematous vasculiti s and hemor­rhage in stroma. The semini ferous tubules demonstrated

566 IND IAN J EXP BIOL, MA Y 2002

Table 1- Effect of Elllblica fruit on Cd induced mortality in ra t

Treatment* mg/kg MOrlalit~+

Cd EIII/JIica No. DeadfTreatcd Percentage

0 0 0/6 0 0 500 0/6 0 3 0 4/6 66 3 500 1/6 16

+Assessed 48 hI' after Cd treatme nt. *Rats were pretreatmcnt with Elllblica fru it j uiee for 8 days, fol­lowed by Cd I hI' after Elllblica on the last day.

significant loss of spermatogenesi s along with charac­teristi c multinucleate st ructures prom inent in the lu­men. Pretreatment with El/lblica showed some cyto­protective effect on Cd-i nduced testicu lar les ions ex­cept that the moderate edema developed in the inter­st itium with minimal hemorrhage and the spermato­genes is appeared less affected, whereas Emblica alone did not produce an y hi stolog ical damage in the tes ti s (Fig. I a,b,c).

In the hepat ic ti ssue, Cd alone produced character­istic foca l necrosis along with acute inflammatory ce ll s. There was congestion at places and sinusoids were not patent. However, when the rats were pre­treated with Emblica, Cd induced hepatic lesions were minimal as some of the hepatocytes at places sti ll showed cloudy swelling and fatty changes. Emblica alone did not influence the hi stology of liver (Fig.2 a,b,c).

Regardi ng th e rena l ti ssue, Cd treatment caused widespread congestion in the cortical region along with prominent tubular necrosis, degeneration and sloughing of tubular epitheli al cells of prox imal and distal tubu les. Emblica pretreatment, had a very mar­ginal protective effect in kidney on the acute tox ic effects of Cd as ev inced by the persistence of moder­ate degree of congestion, degenerati on and desqu ama­tion of tubul ar epithelium with occasional necrosis. The kidney from EJ/lblica alone treated group resem­bled closely to th at of saline control group (Fig. 3a,b,c).

Among the biochemical chan ges, a marked in ­crease in MDA formation in tes ti s, fo ll owed by li ver and kidney was observed in rats treated wi th Cd. EIII­blica pretreatment resulted in a signi ficant decrease in Cd induced MDA. Rats treated with EJ/lblica alone displayed a sli ght lowering in MDA formation in iiver and kidney but not in testi s (Table 2).The total sul f­hydryl content of liver and kid ney was elevated in Cd treated rats. However, on El11blico pretreatment these Cd induced values dec lined substanti all y (Table 2).

;..- . . . .. " . ! • '- .

Fig. I - Microscopic evaluation of tes ti cular ti ssue from cad­

mium chl oride, Elllblic{/ pretreated and Elllblica alonc group.

H & Ex 125. (a) Microscopic changes in rnttestes 6 hr foll owing

admini stration of cadmium chl oride (3 mg Cd/kg, ip). The

seminiferous tu bu les dcmonstrated significant loss of spermJto­

genesis along with multinucleatc structures promi nent in the lu­

men. Interstiti al ti ssucs showcd cdcma, hemorrhage and vasc ular

thrombosis. (b) Section of testcs from rats pretreatcd with Elllblica

(500 mg/kg, orally) for 8 days followed by Cd I hr. aftcr Elllblictl

on the last day. The se miniferous tubu lcs appea red normal, except

that thc edema pers isted in the interstitium with minimal hcmor­

rhage. (c) Sect ion of tcstes fro m rats treated with EllIbl ica alone.

The seminiferous tubu lar ce ll s and interstitialtissuc were normal.

KHA NDELWAL 1'1 al. : CADM IUM TOX ICITY & EM13L/CA OFFICINA LIS 567

The activiti es of serum GOT, GPT and GGT sig­nificantl y increased in rats treated with Cd as com­pared to the untreated group . The va lues of ALP re­mained unaltered. However, in the rats pretreated with

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Fi g. 2- Microscopic cv:ilua ti on of hepati c ti ssue froll1 cadmiu lll chloride Elllblico pretreated and EIIIMica alone group, II & Ex 500. (a) Section of hepat ic tissue 6 hr following admini strt ion or cad­mium chloride (3 mg/kg, ip). Focaillecro~is al ong with inriltrati on or acut e inflammatory cd ls. Congcsti'lll and s inu~o id s were not patcnt. (b) Sect ion or lil'cr from rat pret reated wi th Elllblictl for 8 days, rollowcd by Cd I hr. a rter £lIIb/ica on the last lIay. Hepat ic lesions were minimal. Some! of the hepatocyte:, ,till ~ howcd

cloudy swclling and rally changes . (c) Sec ti on oj' li ver from rat treated with Elllblica alone. No hi stolog ica l changes were l:vidcnt.

ElI1blica followed by Cd, the increased levels of seru m GOT, GPT and GGT was partiall y prevented (Fig. 4). ElIlblica in the present study causc:d sli ght induction or MT in li ve r and MT like protein in testes, simil ar to

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Fig. 3-Microscopic eva lua t:on or ren ,d tissue rrom cadmi um chloride, Elllblim pretreated and Elllblica alonc group. H & E x SUO (a) Sect ion of rc nal conex 6 hI'. followin g ad min istrat ion of C:Jd ­lI1iu m chloride (3 mg Cd/kg. ip). Widespread congcstion in the con ica l regi un along wi th prom inent necrosis. Dege neration and sloughing of proximal nnd dis tal tubu lar cpithcl ial cells. (b) Se,·· tion of rcnal cortex rrom rat pret reated with Elllblim for ~ day~. fo ll owcd by Cd I hI'. aftcr Elllhlim on :hc last day. Persistcnce 01' moderate degrec of l:o ngcstion. degcnci ation and dcsqu:tm:tIllHl or tub ula r epithcl ium with occasional necrosis and presence of cast ill the: lumen. (c) Sect ion of rcnal cortcx from rat treated with ElIl iJlica alone:. GI(lllleruli alld tuhules werc n orlll ~!I.

568 INDIAN J EX P S IOL, MA Y 2002

the inducti on of hepati c MT reported by ascorbi c acid, but to a lesser degree. Cadmium induced MT in li ver and kidney was reduced by 28% and 24% respec­tively by Emblica pretreatment (Tab le 3).

Since ElIlblica clearl y amel iorated the tox icity of cadmium as assessed by leth ality, pathologica l and biochemical changes, ex peri ments were performed to

determine if it was due to altered tox icok ineti cs of Cd. As co mpared to the untreated rats, increase in Cd was observed in li ver fo llowed by kidney and testis. EI1l­blica pretreatment, however, had a marked effect on

the distribution of Cd in these tissues. The uptake of Cd was reduced by 26, 33 and 48% in the hepati c, renal and tes ti cul ar ti ssue, respec ti vely (Table 3).

Discussion The present findings based on the leth ality pattern ,

hi stolog ica l changes in the target organs, Cd and MT levels and various biochemi cal alterati ons in seru m and orga ns indicate that Ernblica pretreatment moder­ated the acute cytotox icity of Cd in rat. Several possi­bilities ex ist as to the mechani sm by whi ch El7lblica

Tabl e 2-Effcct of £lIIblica fru it on Cd induccd lipid pcrox idat ion (MDA) and tota l sulphyd ryl (S I-I ) contcnt in ratti ss ucs

IValucs arc mcan ± SD of fi vc an imal s in cach group] Trcatmcnt* mg/kg

Cd

o o 3 3 F (3. 16)

£1II1)lic((

0

500 0

500

MDA

33.0 ± 2.89

28.3 ± 2.03 42 .6 ± 5.05 b

33.7 ± 2.92)' 12. 17

Livcr Total SI-I

25.4 ± 1.75 28.6 ± 0 .99"

36.9 ± 0.93" 28.4 ± 0.66""

73. 14

Kid nc}'

MDA Total SH

40.2 ± 3.67 22.4 ± 4.45 35 .5 ± 5.67 20.4 ± 2.97

48.8 ± 1.98b 24.9 ± 1.68 39.9 ± 1.72)' 20 .2 ± 3.36£

9.47 3.45

Tcstes

MDA

27.8 ± 7.78

29.9 ± 8.68 58.0 ± 14.49" 33. 1 ± 8.88)'

7.42

Total SH

14.9±2.7 1

19.9 ± 0.73"

14 .8 ± 1.79 20 .1 ± 5.03"-'

8.55

Lipid perox idation was cstimatcd by monitoring malondialdchydc (MDA) formation and cxpressed as n mol MDA/60 min/g tissuc. Total S I-I contcnt was cxpresscd as ~t mol/g ti ssue. "p < 0.00 I, b p< 0.0 I. C I' < 0.05 as co mparcd to un trea ted val ucs.

'I' < 0.00 I, )' 1'< 0.0 I, I. I' < 0.05 as compared to Cd treated va lucs. *Dcta il s in Tablc I

OCd(-) mCd(+)

700 GOT

600 .a

~ 500 :::::>

a. -I 400 c(

GPT ALP

~

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a. 300 [b (!)

J r ~ 0 (!) 200

~ II 100

:::: 0 ;::>

None 500 None 500 None 500

Concentration of Embllca (mglkg)

GGT 5

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t 1

:::: 0 None 500

Fig. 4-EfTect of £lIIblica fruit on Cd induccd GOT, GPT, ALP and GGT in se rulll o f rat. Values arc mcan ± S D o f fivc ani mal s in each group. "p < 0.00 I. hI' < 0.0 I , '"/' < 0.05 as compared to thc untrcatcd values. ' p < 0.00 I, )'P < 0.0 I, /1' < 0.05 as compared to the Cd trcatcd values.

KHANDELWAL el al.: CADM IUM TOX ICITY & £MBLICA OFFlC/NA LIS 569

Table 3-EITcct of' £lIIblica I'ruit on Cd di stribulion and mctall othioncin (MT) Icvels in ral ti sslIcs

I Valu.:s arc mcan ± SD or f'i vc an imals in each group 1

Tre:llmcnt" Illg/kg Cd o o 3 3 F1J .(6)

£lIIblica o

500 o

500

Li ver Cd MT

1.9 ± 0.84 16.4 ± 1.49 1.2 ± 0.58 23.4 ± 5.74

37.9 ± 3.39" 84.9 ± 6.77" 27.7 ± 6.05"·x 61.2 ± 13.9"'"

11 2.0 1 60.49

Cadm ium and MT levcls were cx pressed as pg/g li ssuc. "I' < 0.00 I, "/J < 0.0 I, cp < 0.05 as compared to unt rcatcd va lucs. x/, < 0.00 I, Yp< 0.0 I, I I' < 0.05 as compared to Cd lreated values . " Delai ls in Table I

affo rded protec ti on. ElIIblica pretrea tment reduced the Cd levels in li ver, kidney and tes ti s. The ameliorative ro le of ElIIblica evidenced by a normali zi ng trend in morphological and serum biochemi cal picture strongly suggest that the ElIlblica may exert a stabili zing action on cell membranes. This in turn may affect the leak­age of enzy mes and other metabolic products into the bl ood circulation and may also reduce the up take of Cd by li ver, kidney and tes ti s. The red uced levels of MT in li ver and kidney in the ElIIblica pretreatment group also suggest reduced uptake of the metal in the two organs. The possibility of enh anced metal excre­tion can not be ruled out. On the con trary , the MT like protein in the testi s ex hibiting an increase, propose a different mechan ism of action wi th respect to the amel iorati ve ro le of Embl ica.

The ameli orative potenti al of many vegetables and fr uits is attributed to the combined effect of their con­sr i tuents rather than to a si ngle fac tor for e.g. the an­timutageni c ac tivity of spinach is due to chl orophyllin and ascorbic ac id 13 wh il e in citrus fru its, to citri c ac id and ascorbi c ac i d 2~ .

Since li pid peroxidation is considered an earl y and sensitive index of Cd ex posure25

.26 the lowering of Cd

induced MDA format ion by ElIIblica pretreatment in the present study could be attributed to the inherent antioxidant property of ElIlblica fr uit. Hi gher efficacy of phyllanthus fr uit ex tract rather than of ascorbic acid was reported in allev iating the clastogenic act ion of lead and aluminium in bone marrow cells of mice27.

Some studi es, espec iall y in the case of tes tes, have suggested relati onsh ip between lipid peroxidat ion and CJ . d I hi' . ?X?,) I f' Cd . In uccc emorr 1aglc necros Is - '- . n act, In-duced tes tic ul ar tox icity can be prevented by pre­treatment with an ti oxidants such as ascorbic acid 30,

a -tocopherol3l and methyl B 1232, Thi s indicated that

Kidnc~ Testes Cd MT Cd MT

0.3 ± 0.1 1 22 .0 ± 4.87 0.2 ± 0.08 23 .2 ± 1.26 0.4 ± 0.10 23.2 ± 7.30 0.2±0.1 0 31.7 ± 5.22 8.4 ± 1.43" 32 .0 ± 8.56" 1.2 ± 0.09.1 22 .8 ± 2AI

5.6 ± 0.89"·x 24.3 ± 2.68"·x 0.6 ± 0. 15"'" 37.2±5. 16b.y

90.07 2.06 78.42 5.17

Cd in duced lipid perox idati on plays a causati ve role in testicular les ions. The present study shows that ElIIblica pretreatment reduced th testicul ar MDA formation by 50% as well as the typical spectru m of testi cu lar lesions induced by Cd. In contrast to the tes tes, some studies have indicated that lipid perox i­dation does not seem to playa causative role in Cd induced li ver les ions 20.33. The mechani sm of Cd in ­duced toxicity may differ in different ti ssues or ti ssue components. A modest increase in Cd induced MDA formation and total sulphydryl in live r and kidney were modulated on pretreatment with ElI1bl ica.

The protective effect of L-ascorbi c ac id in pr ve nt­ing Cd induced mortality in mice was related to MT induction in liver34

. Similar mechani sm appears to be operative to some ex tent in the present study. Kid ney, not being the major organ to be affected by a single acute dose of Cd, showed mild morphological altera­ti ons which still persisted wi th El7/b licC/ pretreatment.

The results of thi s study suggesL that the ElIIblicC/ fruit as a whole (compri sing of vitam in C, tann ins, cellulose, amino acids, minera ls, metals, cytokine-like substances etc .) induces endogenous antioxidant de­fence system and reduces lipid peroxidation by Cd in target organs which can have important di rect cyto­protective effects, spec iall y in the event of oxidant stress induced injury by modifying its toxicokinet ics and cell membrane integri ty as well as stimulating MT synthes is. Further experiments are underway to study the therapeuti c efficacy of ElIlblica in long term Cd intoxication in rat.

Acknowledgcmcnt The authors are grateful to Director, Industrial

Toxicology Research Centre, fo r hi s keen interest in thi s work. The authors wi sh to ack nowledge Mr. Ram

570 IND IAN J EXP BIOl, MA Y 2002

Lal and Mr. Ki shan Lal for their assistance in the art work and Mr. R. S. Verma for computer ass istance.

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