April-June 1987Ind. J. Physiol. Pharmac.

eral Manager, IDPL Researchs Boehringer Ingelheim, Westirna and Mr. S. Mallesham for

; of alinidine (N-allyl clonidine,: 199p, 1981.

ifibrillatory properties of alinidine110 : 301-306. 1985.

activity and structure in derivatives

lffects of a new imidazoline ST-375

I. Birkenhager. Comparison bet-,214: 347-350, 1975.

midine (ST-567) a substance withnac., 306 : 255-262, 1979.

nes. Phetmec, Review" 32: 337.

IIVOUS system and the cardiovas-~3,1982.

:ular effects of clonidine and imida-101. Pbermec., 19: 1-10, 1978.

lion of imidazolines IV. Structuralation by clonidine and series of72. 1980.

'nists and antogonists on ouabain-sc., 89 : 385-388, 1986.

I of flutonidine (ST -600) in rat~70-272,1982.

and inhibitory effects of flutoni-uterine smooth muscles. Ind. J.

IS into the effects of clonidine like16: 41-42, 1984.

EFFECT OF LEAD ON ERYTHROPOIETIC SYSTEM OFINTACT AND SPLENECTOMIZED RATS

A. K. GAUTAM AND A. ROY CHOWDHURY

Division of Histochemistry & Cytotoxicology,

National Institute of Occupational Health,

Meghani Nagar, Ahmedabad - 380016

Summary: Erythropoietic alterations in normal and splenectomized mature male rats treatedwith aqueous lead acetate intraperitoneally at dosages of 4 mg/kg and 6 mgjkg body weightwere observed over a period of 30 days. Significant retardation in growth might be due togradual increases in lead toxicity. The elevated blood lead level, increased urinary b -arnino-levulinic acid (ALA-U) excretion, depletion in RBC and haemoglobin content and morenumber of reticul ocvtes in peripheral blood indicated the increased intensity of lead toxicityand inhibitory effect on haem biosynthesis. The accelerating action of lead on erythropoieticcellular series i.e. pronormoblast, early and intermediate normoblast and late normoblast wasevident by the significant increase in number of cellular count both in intact and splenecto-mized rats after treatment with lead.

Key words: splenectomy reticulocyte bone marrow cellsIead acetate

INTRODUCTION

Exposure of lead in industry caused different systemic disorders in human (6,14).Chronic lead exposure induces acute haemolytic crisis, inhibited haem biosynthesis andultimately resulted in anaemia in humans (1, 15). Few studies also confirmed the similareffects after chronic lead exposure in experimental animals (4). Animal studies havealso been reported b -aminolevulinic acid dehydratase inhibition in the tissues concurrentlywith the inhibition in the circulating erythrocytes after 30-40 days treatment of leadacetate. Similar experiment showed the -ultrarnicroscopic changes of mitohondria in ratbone marrow cells in early stages of poisoning (10).

Several pathological disorders were observed after splenectomy, like deficiency inplasma cells, megakaryocytes and bone marrow precursor cells (9)~ But little is knownregarding the effect of lead on erythropoietic processes in mammalian system. Therefore,

118 Gautam and Chowdhury April-June 1987Ind. J. Physiol. Pharmac.

the present investigation was undertaken to determine the relative influence of lead onervthrvpoiesis in intact and splenectomized rats.

MATERIAL AND METHODS

Sixty male albino rats, weighing 135±5 gm were equally divided into six groups(n=10). All groups (Gr. I-VI) were maintained on standard diet and water was suppliedad libitum. Gr. I served as control. Animals of groups 11,IV and VI were anaesthetized bysodium pentabarbitone at a dose of 30 mg/kg/rat (ip) and operated for splenectomy.The rest groups (Gr.11Iand V) were considered for sham operation. Following that ani-mals of all groups were kept for seven days in post operative care. Aqueous solution oflead acetate was administeted at dosages of 4 mg/kg to Gr.1I1 and IV and 6 mg/kg toGr.V and VI intraperitoneallv daily over a period of 30 days. Animals were observedcarefully throughout the experimental period.

Body weight was recorded on every alternate day. Blood samples were collectedon initial day, 15th and 30th day for erythrocyte (RBC) count, haemoglobin (Hb) contentand lead estimation by Atomic Absorption Spectrophotometer (AAS) (13, 5). Bloodsmears were analysed for reticulocyte count alter staining with 1% brilliant cresyl blue(13). Twenty four hour urine samples were collected as per above experimental scheduleand processed for 0 -aminolevulinic acid (ALA) estimation (8). After completion of leadacetate treatment on the 38th day, animals were sacrificed by cervical dislocation. Themarrow samples collected from femur, were processed for bone marrow lead content byAAS and for smear preparation (11). Different developing stages of erythropoietic cellswere counted at 1000x after staining the smears by 1% leishman (13). Student t-testwas employed to determine the level of significance.

RESULTS

Significant decrease in body weight was observed in all experimental groups (Gr.11-VI) in comparison to control over a period of 30 days. Decrement is more significantin both the intact and splenectomized rats treated with 6 mg/kg lead acetate (Gr. V, VI)(Fig.1). Lead content in blood and bone marrow was significantly high in all the treatedgroups (Gr.III-VI) (Figs. 2 and 3). Specially Gr.V and VI exhibited fifty fold increase inblood lead content (P<0.001) whereas five times increase were noted in bone marrow inthe same groups over a period of 30 days (P<0.001). Intermittent observations (0, 15,30 days) of urinary ALA excretion indicated gradual increase in all lead treated groupsboth in intact and splenectomized rats (Gr. Ill-VI). Highly significant increase (P<0.001)was observed in Gr. V and VI on 15th and 30th day (Fig.4).

210.,A

195E0'-s:0' 180~>-

't:J0 165m

150L-

Fig. 1: Changesexposed I

400

_300Eoo•...--0\<-

200

6

3

o

Fig. 2 Elevated blc

April-June 1987lod. J. Physiol. Pharmac.

relative influence of lead on

ually divided into six groupsdiet and water was suppliednd VI were anaesthetized by

operated for splenectomy.ration. FOllowing that ani-care. Aqueous solution ofr.III and IV and 6 mg/kg tos. Animals were observed

od samples were collected•haemoglobin (Hb) contenteter (AAS) (13, 5). Bloodith 1% brilliant cresyl blueove experimental schedule

. After completion of leadcervical dislocation. The

ne marrow lead content byagesof erythropoietic cellsman (13). Student t-test

I experimental groups (Gr.rement is more significantg lead acetate (Gr. V, VI)ntly high in all the treatedited fifty fold increase innoted in bone marrow in

ittent observations (0, 15,in all lead treated groupsieant increase (P<0.001)

2101h-.

195E0\-.C-0\ 180~>-

'C0 165m

150

Con.

oooeoaoo

xX

X

X j

119

oo I2l

o 121

5 p.Con. 'PO 'Sw'Pti4 mg/kq

'Pb"swPb6 mg/kg

Fig. 1 : Changes in body weight over a period of 3 days in lead acetate (i.p.)exposed normal and splenectomized rats.

400

_300Eoo

-.....01a,

200

6

3

o 01n IV V

CONTROL (sPl N. ECTM. 11CONTROL

It mg/kgINTACT 1114mgA<gSpln.Ectm. IV6mg/kg

INTACT V6 mg/kg

VI Spin. Ettm. VIIII

Fig. 2: Elevated blood lead level in lead exposed (i.p.) normal and splenectomized rats.

120 70

5.0Fig. 3: Accumulation of lead in bone marrow of lead exposed normal and splenectomized rats.

i4.0

3.0E02.0o-<,Q\ 1.0E

0.5

0.25

0.0

Volume 31Number 2

CONTROL r:JSPlN. ECTM.

~ 8 IcoCONTROL

. , l""'Ie• 4 mg/kg 'Pb [IJ 0eINTACT v<, 0• 4 mg/k9·p.b' c

~0 4 0:.~ SPLN.ECTM ='E 0

6 mgjkg 'Pb'~

2 0INTACT

06 mg/kg 'Pb' 11 0 aSPlN.ECTM.

Fig. 5 :

f!1CaNT ROL I.!J

4 mg/kg 'Pt> [JJINTACT

~hl~. cc~.~. CONTROL ID4mg/kg Pb Spl n

Ectm.

r

50

E 300'1<,

CTI~

T

10 e<SI

4>

6mg/kg 'Pb INTACT ~

6mg/kg Pb Spin. Ectm.1.

o DAYS 15

(\)

(\)

(\)

30

Fig. 4: Effect of lead acetate (i.p.) treatment on urinary ALA excretionin normal and splenectomized rats.

CON

140

0

0

~12 0

E 11000\

10 0

0

0

Fig. 6: Haemo

Volume 31 Effect of Lead on Erythropoietic System 121Number 2

CONTROL [!JSPlN. ECTM rm 8 ICaNT ROL 11 Spin. Ectm. IkT 1'"1 "IDCONT ROL CONTROL4 mg/kg 'pt) [IJ ""e

INTACT e 0 0v •4 mgjk9 ·P.b'<, 0 0

~c •SPLN.ECTM0 4 00

6 mgjkg 'Pb''f 0 0 •

INTACT §§1 2 0 0 •6 mg/kg 'Pb' 0 0 •li 0SPlN.ECTM. 0 15 30 o 15 30 0 15 30 o 15 30

DAYS DAYS

rmal and splenectomized rats.

Fig. 5: Effect of lead acetate (i.p.) exposure on erythrocyte count innormal and splenectomized rats.

CONTROL

•e00'o 0 •00'00'00,

00"oo •o •

14

12X

E 110\

10

IlSpin. Ectm.

CONTROL

0 !SI •0 ISl •0 IS! •0 IS! •0 !SI

o 1S 30

IV

o 15 30 0 15 30DAYS

o 15 30D,!WS30

ALA excretion Fig. 6: Haemoglobin in normal and splenectomized rats exposed to lead acetate (i.p.I.

122 Gautam and Chowdhury Apri I-J une 1987Ind. J. Physiol. Pharmac.

20 CONTROL [:J Spln.Ectm. CONTROLflli 4mg/kg Po [l]6mg kg 'Pb ~NTACT

INTACT ~ 6rng kg PbiSpl n. Ectm.

4 mg/ kg 'PO1.5 Spl n.Ectrn.

t-e 10o

0.5

o15 30DAYS ~o

Fig. 7: Changes in reticulocytes (%) in lead exposed (i.p.) normal and splenectomized rats.

Hematological stduies were carried out on 0, 15 and 30 day. Significant decreasein RBC numbers and haemoglobin content CHb') was observed after lead acetate treat·ment in intact (Gr.lIl, V) and splenectomized groups (Gr. IV, VI) on 15th and 30th day.Decrement was significant at a level of PLO.05 in RBC count on 30th day in Gr. VandVI (Fig. 5). Highly significant (PLO.OO1) diminution of Hh was observed in Gr.V and VI(Fig.6). Gradual increase in reticulocyte numbers in peripheral blood was observed inall treated groups (Gr. Ill-VI) on 15 and 30 day in comparison to control and splenecto-mized control (Fig. 7). Ervthropoietic cellular count in bone marrow observed after 30days treatment indicated graded increase (PLO.05) in number of different cells i.e.pronormoblast (PNB), early and intermediate normoblast (ENB and INB) and late normo-blast (LNB) in all experimental groups (Gr. II-VI) (Fig. 8).

DISCUSSION

Retardation in growth was due to lead toxicity as it was evident by the siqnificantincrease in blood lead content and urinary ALA excretion (12). Higher accumulationof lead in bone marrow in all lead treated groups revealed the deposition tendency of themetal in bone. Heavy metals conjugate with specific proteins in blood and form a

Volume 31Number 2

403020

L NB

10L..65::J

~52~

~39~26

ENB&

d INB

~ 13QJu

15

9PNB

3•

Fig. 8: Effectcell c

stable bonds which areain RBC numbers andHnumbers in peripheral blocin turn inhibited the haemin both intact and spleneccauses low 'Hb' concentrIn the present investigatupto the stages of late nehaematological disturbancthat spleen did not havemay be suggested that thlnot have any difference fr

April-June 1987Ind. J. Physiol. Pharmac.

4mg/kg 'PO [J]INTACT

b

5 ~ 30al and splenectomized rats.

o day. Significant decreaserved after lead acetate treat-V, VI) on 15th and 30th day.nt on 30th day in Gr. Vandwasooserved in Gr.V and VIheral blood was observed inn to control and splenecto-ne marrow observed after 30umber of different cells i.e.B and INB) and late normo-

asevident by the significant(12). Higher accumulation

e deposition tendency of theoteins in blood and form a

Effect of Lead on Erythropoietic System 123

~CONTROL I t!ISPLN. ECTM. ffi

CONTROL IT!#

4mgfkg 'Pb rmINTACT mUll

4mg/kg'pb'TIlfBSpin. Ectm. 11

6mg/kg 'Po ~INTACT V ~

6mg/kg'Pb ISpln.Ectm.

Fig. 8: Effect of lead acetate exposure (i.p.) on bone marrow erythropoieticcell count in normal and splenectomized rats.

stable bonds which are actively get associated within the tissues (2). Gradual decreasein RBC numbers and 'Hb' content on 15th and 30th day alongwith elevated reticulocytenumbersin peripheral blood in lead treated rats indicated the potential lead toxicity whichinturn inhibited the haem biosynthesis after lead treatment (7). These changes are similarin both intact and splenectomized rats treated with different dosages of lead. Splenectomycauseslow 'Hb' concentration and induces the hyper function of bone marrow cells (3).In the present investigation, the mitotic accelerating action of lead on erythropoiesisupto the stages of late normoblast was well evident in all experimental groups. Thehaematological disturbances and erythropoietic changes induced by lead in rats, suggestedthat spleen did not have an evident role in the process of lead toxicity. Therefore, itmaybe suggested that the intensity of lead toxicity in intact and splenectomized rats didnot have any difference from the present experimental conditions.

, 24 Gautam and Chowdhurv April-June 1987Ind. J. Physiol. Pharmac.

ACKNOWLEDGEMENTS

The authors are thankful to Dr. S. K. Kashyap, Director NIOH, Ahmedabad for hisencouragements. Thanks are also due to Mrs. H. S. Trivedi, Miss B. M. Shah andMr. Y. V. Vaghasia for their technical assisstance.

REFERENCES

1. Berk. P.D., D.P. Tschudy. L.A. Shepley. J.G. Waggoner and N./. Berlin. Hemotologic and bioche-mical studies in a case of lead poisoning. Am. J. Med.,: 48: 137-144, 1970.

2. Cherian, M.G. and M. Nordberg. Cellular adaptation in metal toxicology and metallothionein.Toxicoloqv., : 28: 1-15, 1983.

3. Crosby, W.H. Hypersplenism. In 'Hematology' edited by Williams, W.J. et al. Mcgraw Hill BookCo. 2nd ed. New York, p. 611-617, 1977.

4. De Bruin, A. Certain biological effects of lead upon the animal organism. Arch. Envlron. Health23 : 249-264, 1971.

5. Delves, H.T. A micro sampling method for the rapid determinatin of lead in blood by atomic absorp-tion spectrophotometry. Analyst, 95: 431 -438. 1970.

6. Eyden, B.P., J.R. Maisin and G. Mattelin. Long term effects of dietary lead acetate on survival.body weight and seminal cytology in mice. Bull. Environ. Contem, Toxicot .• 19 : 266-272, 1978.

7. Fujii, H. A role of red cell pyrimidine 5' -nucleotidase in experimental lead poisoning. Arch. Toxicol ..43: 59·64, 1979.

8. Grabecki, J., J. Haduch and Urbanwiex. Die emifachen Bestimmungem ethodan der b -eminolevuhmcSOUTein Harm. Int. Arch. Gewebepathol. Gewebehyg .. 23 : 220-224, 1967.

9. Koch. J.L. Aplastic anaemia and splenectomy. Arch. Intern. Med., 119 : 305·310. 1967.

10. Millar. J.A., V. Battistini, R.L.C. Cumming. F. Carswell and A. Goldberg. Lead and b -arninolavu-linic acid dehydratase levels in mentally retarded children and in lead poisoned suckling rats. Lancet ..2 : 695-698, 1970.

11. Mylroe, A.A., L. Moore and U. Erogbigbo. Influence of dietary factors on blood and tissue leadconcentration and toxicology. Toxicol. Appl. Pharmacol .. 41 : 361-377, 1977.

12. Roy Chowdhury, A .• A. Dewan and D.N. Gandhi. Toxic effect of lead on testis of rat. Biomed.Biochim.Acta .. 43: 95-100.1984.

13. Seiverd. C.E. Hematology for medical technologists. 4th ed. Lea and Febiger, Philadelphia, 1972.

14. Tsuchiya, K. Lead, In 'Hand book on the Toxicology of metals', edited by Friberg, L. et et., Elsevier-North Holland Biomedical Press, p, 451-484, 1979.

15. Urbanowicz. H. Occupational exposure to inorganic compounds of lead. Arch. Environ. Health.23: 284-288, 1971.

A.CTIO OF SODIAMINI

O. P. SETHI*, A. M. ISM

Govt

Summary: Five of thvarious eNS attributesanalgesics in variousactivity of mice and poMES were not pronounring ED60and LD50in m

Key words: ethylenedether nar

Actions of five subof dog, on skeletal and SI

some of their actions on

Male or female alof 5. All drugs were dis

Present address: -Depart"Hamd+Direct