Effects of prenatal and neonatal exposure to lead on white blood ...

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Effects of prenatal and neonatal exposure to lead on white blood ...

Journal ong>ofong> Cell ong>andong> Molecular Biology 10(1):33-40, 2012 Research Article 33

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

ong>Effectsong> ong>ofong> ong>prenatalong> ong>andong> ong>neonatalong> ong>exposureong> ong>toong> ong>leadong> on white blood

cells in Swiss mice

Ragini SHARMA*, Khushbu PANWAR, Sheetal MOGRA

Environmental ong>andong> Developmental Toxicology Research Lab, Department ong>ofong> Zoology, M. L. S. University,

Udaipur- 313001 Rajasthan, India

(* author for correspondence; taurasragini@yahoo.com)

Received: 27 August 2011; Accepted: 25 May 2012

Abstract

Lead ong>exposureong> is one ong>ofong> the major environmental issues for children ong>andong> women ong>ofong> child bearing age. It

crosses the placental barrier ong>andong> its greater intestinal absorption in fetus results in developmental defects.

Lead, as one ong>ofong> the environmental pollutants, can threat the lives ong>ofong> animals ong>andong> human beings in many ways;

especially during developing stages. The present study was carried out ong>toong> study the alterations in different

types ong>ofong> white blood cells (WBC) due ong>toong> chronic ong>leadong> acetate ong>toong>xicity in neonates, which passes from adult

pregnant female during gestation ong>andong> lactation. Lead acetate was administered orally at 8, 16, 32 mg /kg/BW

ong>toong> pregnant Swiss mice from 10th day ong>ofong> gestation ong>toong> 21th day ong>ofong> lactation. Hemaong>toong>pathological ong>andong>

numerical alterations in the WBCs were examined in the neonates after birth at postnatal days 1, 7, 14 ong>andong>

21. Blood smears examined illustrate that ong>leadong> induces disturbances in the development ong>ofong> different types ong>ofong>

WBCs during postnatal development ong>andong> ong>leadong> ong>toong> an abrupt neutrophilic degeneration, immature cells,

abnormal neutrophils, reactive ong>andong> plasmacyong>toong>id lymphocytes. The results ong>ofong> the present study emphasize

that ong>prenatalong> ong>leadong> ong>exposureong> is extremely dangerous ong>toong> developing fetus.

Keywords: Lead acetate, Swiss albino mice, ong>prenatalong>, ong>neonatalong>, white blood cells.

Swiss farelerde ong>prenatalong> ve yenidoğan kurşun maruziyetinin beyaz kan hücreleri üzerine

etkileri

Özet

Kurşun maruziyeti çocuklar ve çocuk doğurma çağındaki kadınlar için majör çevresel konulardan birisidir.

Plasental bariyeri geçer ve fetusta barsaklardan emilimi, gelişimsel defektlerle sonuçlanır. Çevresel

kirliliklerden biri olan kurşun, birçok yönden özellikle gelişim çağı boyunca insan ve hayvan hayatını tehdit

etmektedir. Bu çalışma, yetişkin hamile dişilerden gebelik ve laktasyon süresince yenidoğanlara geçen

kurşun asetatın yarattığı kronik ong>toong>ksisite sebebiyle lökositlerin farklı tiplerindeki değişimleri incelemek için

yapılmıştır. Kurşun asetat, hamile Swiss farelere gebeliğin 10. gününden laktasyonun 21. gününe kadar ağız

yoluyla 8, 16, 32 mg /kg//BW şeklinde uygulanmıştır. Yenidoğanlarda doğumdan sonraki 1, 7, 14 ve 21.

günlerde beyaz kan hücrelerindeki hemaong>toong>paong>toong>lojik ve sayısal değişiklikler incelenmiştir. Kurşunun doğum

sonrası gelişim sırasında lökositlerin farklı tiplerinin gelişimindeki bozuklukları indüklediğini ve ani

nötrong>ofong>ilik olgunlaşmamış dejenerasyona, hücreler, anormal nötrong>ofong>illere, reaktif ve plazmasiong>toong>id lenfositlere

neden olduğunu göstermek için kan yaymaları incelenmiştir. Bu çalışmanın sonuçları ong>prenatalong> kurşun

maruziyetinin gelişen fetus için son derece tehlikeli olduğunu vurgulamaktadır.

Anahtar kelimeler: Kurşun asetat, Swiss albino fare, ong>prenatalong>, yenidoğan, lökosit.


34Ragini SHARMA et al.

Introduction

Lead has been recognized as a biological ong>toong>xicant

ong>andong> different doses have been used ong>toong> study ong>leadong>induced

alterations Prenatal ong>exposureong> ong>toong> ong>leadong>

produces ong>toong>xic effects in the human fetus, including

increased risk ong>ofong> preterm delivery, low birth

weight, ong>andong> impaired mental development; because

during the period ong>ofong> early organogenesis the onset

ong>ofong> greatest susceptibility ong>toong> teraong>toong>genesis occurs

(Falcon et al., 2003). This highly sensitive or

critical period is the time during which a small dose

ong>ofong> a teraong>toong>gen produces high percentage ong>ofong> fetuses

that exhibit malformations ong>ofong> the organ in question

(Wilson, 1973; Desesso et al., 1996).

Pregnancy ong>andong> breastfeeding can cause a state

ong>ofong> physiological stress that increases bone turnover

ong>ofong> ong>leadong>. Lead song>toong>red in the bone moves inong>toong> the

blood, increasing the mother’s blood ong>leadong> level ong>andong>

passing ong>toong> the fetus, affecting fetal development.

Lead is tightly bound ong>toong> red blood cells, enhancing

transfer from maternal circulation through the

placenta ong>toong> the fetus. Fetus is more sensitive ong>toong> ong>leadong>

because the fetal blood-brain barrier is more

permeable. The ong>toong>xic effects ong>ofong> ong>leadong> on blood

indices are well known.

Lead potentially induces oxidative stress ong>andong>

evidence is accumulating ong>toong> support the role ong>ofong>

oxidative stress in the pathophysiology ong>ofong> ong>leadong>

ong>toong>xicity. Lead is capable ong>ofong> inducing oxidative

damage ong>toong> brain, heart, kidneys, ong>andong> reproductive

organs. The mechanisms for ong>leadong>-induced oxidative

stress include the effects ong>ofong> ong>leadong> on membranes,

DNA, ong>andong> antioxidant defense systems ong>ofong> cells

(Ahamed ong>andong> Siddiqui, 2007). Lead interferes with

a variety ong>ofong> body processes ong>andong> is ong>toong>xic ong>toong> the body

systems including cardiovascular, reproductive,

hemaong>toong>poietic, gastrointestinal ong>andong> nervous systems

(Kosnett, 2006), renal functions (Paong>toong>cka ong>andong>

Cerny, 2003) ong>andong> release ong>ofong> glutamate (Xu et al.,

2006). It affects the hemaong>toong>logical system even at

concentrations below 10μg/dl (ATSDR, 2005).

Many reports are available regarding ong>leadong>

ong>toong>xicity ong>andong> its deleterious effects in various

species ong>ofong> animals ong>andong> there has been lot ong>ofong> work

carried out on pharmacokinetics ong>andong> genoong>toong>xicity

but very few researchers tried ong>toong> correlate

haemaong>toong>pathological alterations ong>ofong> ong>leadong> acetate in

different white blood cells at different dose levels

in laboraong>toong>ry animals, especially in mice.

Therefore the current study was performed ong>toong>

clarify the ong>leadong> induced hemaong>toong>logical changes,

especially those related ong>toong> white blood cells, during

gestational ong>andong> lactational ong>exposureong> ong>toong> ong>leadong> in

Swiss mice.

Materials ong>andong> methods

Sexually mature rong>andong>om bred Swiss mice with the

age ong>ofong> 5-6 weeks, weighing 25-30 gm was used for

this study. During the entire experimental period,

the animals were fed on a stong>andong>ard diet ong>andong> water

ad libitum. Mice were kept in the ratio ong>ofong> 1:4 males

ong>andong> females, respectively, ong>andong> females showing

vaginal plugs were separated in the control ong>andong> ong>leadong>

treated group. Lead acetate solution was prepared

by dissolving 4gm ong>leadong> acetate in 12ml distilled

water. Pregnant Swiss mice were given ong>leadong> acetate

at a concentration ong>ofong> 8, 16 ong>andong> 32 mg (266.66,

533.33, ong>andong> 1066.66 mg/kg/bodyweight) from 10 th

day ong>ofong> gestation ong>toong> 21 st day ong>ofong> lactation. Blood

samples were obtained from the tail ong>ofong> pups from

each litter at days 1,7,14 ong>andong> 21 day after birth. The

tip ong>ofong> the tail was cleaned with spirit before being

cut with a sharp blade ong>andong> was not squeezed ong>toong>

avoid dilution ong>ofong> blood by tissue fluid.

Blood cells were studied in smears prepared by

spreading a drop ong>ofong> blood thinly over a clean ong>andong>

sterilized microscopic slide with the help ong>ofong> another

slide moved over the first at the angle ong>ofong> 45ᵒ after

discarding first drop ong>ofong> blood. These blood films

were air-dried ong>andong> fixed in absolute methanol for

15 minutes by dipping the film briefly in a Coplin

jar containing absolute methanol. After fixation the

slides were removed ong>andong> air-dried. Afterward blood

smears were stained with freshly made Giemsa

stain diluted with water buffered ong>toong> pH 6.8 or 7.0

(1:9) stain ong>andong> buffer respectively. The slides were

washed by briefly dipping the slide in ong>andong> out ong>ofong> a

Coplin jar ong>ofong> buffered water ong>andong> air dried again for

taking observations. The erythrocytes appear pink

ong>toong> purple, whereas leukocytes turned blue black in

color. All the experimental work was approved by

the Institutional Animal Ethics Committee.

No./CS/Res/07/759.

Group 1- Control (distilled water only).

Group 2- Exposure ong>toong> 8 mg ong>leadong> acetate (266.66

mg/kg BW) from 10th day ong>ofong> gestation up ong>toong> 21st

day ong>ofong> lactation.

Group 3- Exposure ong>toong> 16 mg ong>leadong> acetate

(533.33 mg/kg BW) from 10th day ong>ofong> gestation up

ong>toong> 21st day ong>ofong> lactation.

Group 4- Exposure ong>toong> 32 mg ong>leadong> acetate

(1066.66 mg/kg BW) from 10th day ong>ofong> gestation up

ong>toong> 21st day ong>ofong> lactation.

The statistical analysis was performed following

t-test for the comparison ong>ofong> data between different

experimental groups. The data was calculated using


prism song>ofong>tware ong>toong> calculate the p values. 100 WBC

from each group were counted at different weeks,

different cell types were identified ong>andong> % ratio was

calculated. For numerical observation highest dose

level was selected.

Results

In the control group all the WBCs showed normal

appearance. The neutrophils in control group were

examined by a very characteristic nucleus with

condensed chromatin. It is divided inong>toong> 3-5 lobes

(Fig.1A, 1, 2, 5 ong>andong> 6) at birth which was observed

with an increase by 5 ong>toong> 6 lobes (Fig.1B, 1 ong>andong> 3) at

the termination ong>ofong> lactation, connected by thin

strong>andong>s ong>ofong> chromatin. Lymphocytes were round or

ovoid at the time ong>ofong> birth (Fig. 1A, 3 ong>andong> 4) but

further on they were found notched or slightly

indented (Fig. 1B, 5 ong>andong> 6). The chromatin was

generally diffusely dense. Ordinarily, nucleoli were

not visible. A perinuclear clear zone surrounding

the nucleus was visible after first week ong>ofong> lactation

in some cells. The cyong>toong>plasm stained light blue ong>andong>

ranges from sparse ong>toong> moderately abundant in

amount. The monocyte in control group were round

with smooth margins, the nucleus was oval,

indented ong>andong> slightly folded (Fig.1B, 4). The

chromatin material was moderately clumped ong>andong>

relatively less dense compared ong>toong> that ong>ofong>

neutrophils or lymphocytes. There was no visible

nucleolus with abundant cyong>toong>plasm.

The administration ong>ofong> ong>leadong> acetate altered the

appearance ong>andong> caused structural changes. The

following hemaong>toong>logical observations were taken

during postnatal period from birth till the

termination ong>ofong> the lactation period upon ong>exposureong>

ong>ofong> different doses ong>ofong> ong>leadong> acetate:

1. At the time ong>ofong> birth (PND1)

Abnormal neutrophils: In ong>leadong> treated groups

the neutrophils showed structural abnormalities in

their nucleus including improper segmentation ong>andong>

lesser condensation ong>ofong> nucleus. At a lower dose the

chromatin material was condensed, all the lobes

were interconnected with each other ong>andong> form a

nodule like structure at one side (Fig.1C, 1).

Degeneration: In ong>leadong> treated group most ong>ofong> the

neutrophils appeared in degenerating state in which

the chromatin material was very less condensed,

fused ong>andong> there was no sign ong>ofong> clear lobulization

ong>andong> segmentation (Fig. 1C, 2).

Immature cells: In ong>leadong> treated group the

number ong>ofong> immature cells was increased (Fig. 1C,

3).

Prenatal ong>andong> ong>neonatalong> ong>exposureong> ong>toong> ong>leadong> 35

Ring shaped: In ong>leadong> treated groups, some

neutrophils showed abnormal ring like appearance

ong>andong> diffuse chromatin material, with unclear

cyong>toong>plasm. In 32 mg ong>leadong> treated group

vacuolization in chromatin material was also

observed ((Fig. 1C, 4).).

Lymphocyte: Reactive (Fig. 1C: 5 ong>andong> 6) ong>andong>

cleaved (Fig. C, 5) types ong>ofong> lymphocytes were

observed in ong>leadong> treated groups.

Monocytes: At postnatal day 1 we cannot

identify any structural change in shape ong>andong> size ong>ofong>

monocyte as observed on postnatal day 21.

2. During first ong>andong> second week ong>ofong> postnatal period

(PND7&14)

The following observations were taken at first ong>toong>

second week after birth:

Degenerated neutrophils: In ong>leadong> treated group

overall numbers ong>ofong> neutrophils were increased

particularly with degenerated neutrophils, however,

their number was less than postnatal day 1. In 16

mg ong>leadong> group on postnatal day 7 the nuclear

material ong>ofong> neutrophil was less condensed ong>andong>

nucleus was divided inong>toong> 2-3 unequal lobes. The

cyong>toong>plasm ong>ofong> neutrophil appeared colorless. At the

dose ong>ofong> 32mg ong>leadong> at postnatal day 7, this severity

ong>ofong> degeneration was very much increased so that

the lobes were broken inong>toong> many small fragments.

No sign ong>ofong> lobulization ong>andong> appropriate

segmentation ong>ofong> neutrophils were found (Fig. 1D,

1).

Ring shaped neutrophils: In contrast ong>toong>

postnatal day 1, ring like nucleus was not observed

in ong>leadong> treated group at postnatal day 7.

Different types ong>ofong> neutrophils: At higher dose

32 mg ong>leadong> treated groups apopong>toong>tic or necrotic

neutrophils were more prominent. These

neutrophils were characterized by 3-4 separate ong>andong>

equal lobes with less condensed chromatin ong>andong>

diffuse cyong>toong>plasmic region (Fig. 1D, 2).

Immature cells: Review ong>ofong> the ong>leadong> treated

smear revealed that most ong>ofong> the leukocytes were

myelocytes, bong>andong>s, myeloblast ong>andong> other immature

ong>andong> unidentified white blood cells with left shift in

leucocytes. A left shift is an increase in the number

ong>ofong> bong>andong> neutrophils ong>andong> other immature cell ong>ofong> the

granulocytic lineage in the peripheral blood (Fig.

1D, 3).

Various lymphocytes: Administration ong>ofong> ong>leadong>

acetate produced great variation in lymphocyte

structurally as well as numerically. Various types ong>ofong>

lymphocytes such as plasmacyong>toong>id, reactive, oval,

irregular, binucleated ong>andong> cleaved lymphocytes

were identified, whereas only reactive ong>andong> cleaved


36Ragini SHARMA et al.

lymphocytes were seen in postnatal day 1,

exclusively in ong>leadong> treated group.

Lead treated group with 16 mg ong>leadong> acetate

produced large lymphocytes ong>andong> most ong>ofong> the

lymphocytes were having irregular; clumpy ong>andong>

smudgy chromatin material with very dense

nucleus (Fig. 1D, 4). The cyong>toong>plasm appeared

completely absent as the nucleus reached its largest

size ong>andong> covered all the cyong>toong>plasmic area. Overall,

number ong>ofong> lymphocytes decreased in most ong>ofong> the

groups. At higher dose (32 mg ong>leadong>) the

plasmacyong>toong>id lymphocytes (eccentric nucleus ong>andong>

intensely blue / basophilic cyong>toong>plasm) (Fig. 1D, 6)

ong>andong> reactive lymphocytes were observed. Reactive

lymphocyte was characterized by relatively very

large, irregular but flattened nucleus with fine

chromatin ong>andong> agranular light blue stained

cyong>toong>plasm (Fig. 1D, 5).

3. At the end ong>ofong> lactation period (PND21)

Abnormal nuclear segmentation: It includes

abnormal segmentation ong>ofong> nucleus, in which the

nuclear lobes were connected with each other. It

gave abnormal appearance ong>ofong> nucleus ong>andong>

chromatin condensation in most ong>ofong> the neutrophils

(Fig. 1E, 1).

Degeneration: In lower doses ong>ofong> ong>leadong> diffuse

appearance ong>ofong> chromatin material was observed in

neutrophils ong>andong> the lobes were fused with each

other as any segmentation was not observed,

whereas in higher ong>leadong> treated group the neutrophils

presented fragmented chromatin material ong>andong> very

less condensation ong>ofong> nucleus which finally ong>leadong>s ong>toong>

cell lysis (Fig. 1E, 2). The nuclear arrangement was

disong>toong>rted, as appear that all the lobes were

intermingled with each other ong>andong> in some cases

form a nodule at one side known as sessile nodule

appeared like hypersegmentation (Fig. 1E, 3).

Immature cells: In ong>leadong> treated group the

numbers ong>ofong> immature cells were increased. A left

shift i.e. presence ong>ofong> immature neutrophils, bong>andong>s,

metamyelocytes, myelocytes ong>andong> other unidentified

immature cells were observed (Fig. 1F, 1 ong>toong> 6).

Lymphocytes: As the dose level increased the

number ong>ofong> lymphocytes decreased. In higher dose

ong>leadong> treated group the lymphocyte appeared large in

size with higher volume ong>ofong> cyong>toong>plasm. The shape ong>ofong>

the nucleus also vary from round ong>toong> elliptical in

structure, termed as reactive lymphocyte (Fig. 1E,

4). Some lymphocytes transformed inong>toong>

plasmocyong>toong>id lymphocyte in which the lymphocyte

contains basophilic cyong>toong>plasm ong>andong> eccentric

nucleus (Fig. 1E, 5).

Monocytes: In ong>leadong> treated groups the shape ong>andong>

structure ong>ofong> the monocyte were modified ong>andong> the

shape ong>ofong> the nucleus was also altered from the

normal reniform (kidney shaped) nucleus. The

indentation ong>ofong> the nucleus became larger ong>andong>

deeper from periphery ong>toong> center. At higher dose

level intensity ong>ofong> the indentation was increased so

that the normal range ong>ofong> nucleo-cyong>toong>plasmic ratio

was disturbed (Fig. 1E, 6). Numerical changes in

different types ong>ofong> WBC ong>andong> percent variations in

different types are incorporated in Table 1 ong>andong> 2

respectively. In present investigation, after

evaluating all the cell types, we can conclude that

ong>leadong> acetate at PND 1 ong>andong> 14 caused significant

increase in number ong>ofong> neutrophils ong>andong> decrease in

lymphocytes, while there was no significant

difference in the number ong>ofong> neutrophils ong>andong>

lymphocytes at PND 7 ong>andong> 21.

Table 1. Various types ong>ofong> WBCs at different postnatal days treated with ong>leadong> acetate.

Groups Neutrophils Lymphocytes Monocytes

Control at PND 1 59.25±1.70 38.5±1.29 2.25±1.70

Lead acetate at PND1 66.00±2.16** 28.25±2.06** 5.75±1.70*

Control at PND 7 57.75±2.21 41.75±2.21 0.75±0.95

Lead acetate at PND7 61.75±3.5 37.5±2.88 0.75±0.95

Control at PND 14 55.25±3.40 44.5±3.10 0.25±0.5

Lead acetate at PND14 61.25±1.70** 37.75±1.70** 1.00±0.81

Control at PND 21 47.75±2.5 47.75±2.21 4.5±2.38

Lead acetate at PND21 52.25±4.57 44.00±2.26 3.75±2.75

Values were expressed as means ± S.D.; 4 animals /group;*=p


Prenatal ong>andong> ong>neonatalong> ong>exposureong> ong>toong> ong>leadong> 37

Table 2. Percent variation in different types ong>ofong> WBCs in ong>leadong> treated groups

Lead acetate at PND1

Lead acetate at PND7

Lead acetate at PND14

Lead acetate at PND21

Neutrophils Lymphocytes Monocytes

Normal 12.3%

Degenerated 12.1%

Ring shaped 8.2%

Immature 4.1%

Abnormal 10.4%

Normal 12.6%

Degenerated 8.4%

Abnormal 16.8%

Immature 10.8%

Ring shaped 6%

Normal 19.2%

Degenerated 19%

Abnormal 9.6%

Immature 6.4%

Apopong>toong>tic 6.4%

Normal 6.18%

Degenerated18.5%

Ring shaped 1.5%

Abnormal 17%

Immature 7.4%

Normal 13%

Reactive 17.33%

Cleaved 8.6%

Normal 12.2%

Plasmacyong>toong>id 7.4%

Reactive 9.8%

Binucleated 2.4%

Large 4.2%

Normal 4%

Plasmacyong>toong>id 4%

Reactive 12.3%

Binucleated 8.2%

Large 4%

Irregular 2%

Oval 2%

Normal 10%

Abnormal 16.4%

Plasmacyong>toong>id1.4%

Reactive 4.4%

Large 5.8%

Irregular 5.86%

Normal 2.0%

Abnormal 3.7%

Abnormal 0.75%

Abnormal 1%

Normal 2%

Abnormal 1.7%

Figure 1. A: Peripheral blood smear ong>ofong> control group showing neutrophil (1-2), lymphocytes (3-4), at the time ong>ofong> birth,

neutrophils (5-6) during second ong>andong> third week ong>ofong> lactation. B: Control group showing neutrophil (1), lymphocyte (2),

during second ong>andong> third week ong>ofong> lactation, ong>andong>, neutrophil (3), lymphocytes (4-5) ong>andong> monocyte (6) at the termination ong>ofong>

lactation. C: Peripheral blood smear ong>ofong> ong>leadong> treated group showing abnormal neutrophil (1), degenerated neutrophil (2),

immature cell (3), ring like neutrophil (4), 5 – cleaved (upper WBC) (5) ong>andong> reactive (lower WBC) lymphocyte (5 ong>andong> 6)

at the time ong>ofong> birth. D: Lead treated group showing degenerated neutrophil (1), necrotic (2), immature cell (3), large

lymphocyte (4), reactive (5) ong>andong> plasmacyong>toong>id lymphocyte (6) During first ong>andong> second week ong>ofong> postnatal period. E: Lead

treated group showing - abnormal neutrophil (1), degenerated neutrophil (2), hypersegmented neutrophil (3), reactive

lymphocyte (4), plasmacyong>toong>id lymphocyte (5) ong>andong> reactive monocyte (6) at the termination ong>ofong> lactation. F: Lead treated

group showing different immature cells at the termination ong>ofong> lactation (1- 6). (All Giemsa stain, 450x).


38Ragini SHARMA et al.

Discussion

Changes in leukocyte parameters are ong>ofong>ten one ong>ofong>

the hallmarks ong>ofong> infection. These include changes

in number ong>andong> in cellular morphology. Review ong>ofong>

the peripheral blood smear can provide significant

insight inong>toong> the possible presence ong>ofong> infection. Early

changes during infection may include an increase in

the number ong>ofong> bong>andong>s, even before the development

ong>ofong> leukocyong>toong>sis. A great shift ong>toong> immaturity (left

shift) may occur when infection is severe, with

metamylocytes or even earlier forms present on the

peripheral blood smear. There are many evidences

ong>ofong> studies conducted on adults ong>andong> RBC concerning

ong>leadong> ong>toong>xicity, but very few reports are available

regarding haemaong>toong>pathological alterations ong>ofong> ong>leadong>

acetate in different white blood cells. Significant

decrease in RBC count, hemaong>toong>crit (Hct) ong>andong>

hemoglobin (Hb) were seen in rats ong>andong> human with

high blood ong>leadong> levels. (Alexa et al., 2002; Noori et

al., 2003; Othman et al., 2004; Toplan et al., 2004)

In our study the control groups showed all the

leukocytes in normal appearance. Still some altered

types ong>ofong> WBCs were also observed. The

administration ong>ofong> ong>leadong> acetate alters the structure

ong>andong> number ong>ofong> WBCs. The nuclear arrangement

was also disong>toong>rted. In ong>leadong> treated groups the shape

ong>andong> structure ong>ofong> the monocyte was also altered with

reniform (kidney shaped) nucleus. At higher dose

level this intensity ong>ofong> indentation was increased so

that the normal range ong>ofong> nucleo-cyong>toong>plasmic ratio is

disturbed ong>andong> appeared as reactive monocytes. Our

findings are also in support ong>ofong> DeNicola et al.

(1991) with the evidence ong>ofong> reactive monocytes

enclosing the cyong>toong>plasm became more intensely

basophilic ong>andong> vacuolated. This usually indicates a

chronic inflammaong>toong>ry process or may be seen with

hemoplasmas in the cat.

Toxicity in neutrophils is defined by the

presence ong>ofong> Döhle bodies (small, basophilic

aggregates ong>ofong> RNA in the cyong>toong>plasm), diffuse

cyong>toong>plasmic basophilia etc. In our study each ong>leadong>

treated group in ong>neonatalong> period, represents

increased number ong>ofong> degenerated neutrophils

particularly at birth. In the 16 mg ong>leadong> exposed

group, during first week ong>ofong> lactation, the nuclear

material ong>ofong> cell was less condensed ong>andong> nucleus

was divided inong>toong> 2-3 unequal lobes with colorless

cyong>toong>plasm. At higher dose ong>ofong> 32 mg ong>leadong>, this

severity ong>ofong> degeneration was very much increased

with many small fragments ong>ofong> nuclear material ong>andong>

no sign ong>ofong> lobulization ong>andong> appropriate

segmentation ong>ofong> neutrophils were observed.

In a study performed on young dogs,

development ong>ofong> anemia, leukocyong>toong>sis,

monocyong>toong>penia, polychromaong>toong>-philia, glycosuria,

increased serum urobilinogen, ong>andong> hematuria has

been reported (Zook, 1972). Lead suppresses bone

marrow hemaong>toong>poiesis, probably through its

interaction with the enteric iron absorption (Klader,

1779; Chnielnika, 1994). In some reports,

leukocyong>toong>sis has been attributed ong>toong> the ong>leadong>-induced

inflammation (Yagminas et al., 1990).

Hogan ong>andong> Adams, (1979) reported a threefold

increase in neutrophil ong>andong> monocyte count along

with severe leukocyong>toong>sis in the young rats that were

exposed ong>toong> ong>leadong>. The present investigation revealed

that administration ong>ofong> ong>leadong> acetate alters the

appearance ong>andong> cause structural changes. The

nuclear arrangement was disong>toong>rted with

intermingled lobes ong>andong> in some cases formed a

sessile nodule.

Controversies exist about monocytes; since in

some studies ong>leadong>-induced monocyong>toong>penia

(Xintaras, 1992) ong>andong> in others significant increases

in monocyte count have been reported (Yagminas

et al., 1990). The reason for such difference is

probably due ong>toong> the extent ong>ofong> ong>leadong>-induced

inflammation.

Mugahi et al. (2003) investigated additional

hemaong>toong>ong>toong>xic effects ong>ofong> ong>leadong> on the erythroid cell

lineage ong>andong> leukocytes following long-term

ong>exposureong> in rats. Wahab et al. (2010) showed that

ong>leadong> caused a significant decrease in hemaong>toong>crit,

RBC, WBC, hemoglobin concentration, mean

corpuscular hemoglobin, mean corpuscular

hemoglobin concentration ong>andong> lymphocyte ong>andong>

monocyte count; ong>andong> significant increase in

neutrophil count. The results ong>ofong> the present study

are also parallel ong>toong> the above findings. In ong>leadong>

exposed pups there was significant increase in the

number ong>ofong> neutrophils at different weeks after birth,

but decrease in the number ong>ofong> lymphocytes. The

shortened life span ong>ofong> erythrocytes is due ong>toong>

increased fragility ong>ofong> the blood cell membrane ong>andong>

reduced hemoglobin production is due ong>toong> decreased

levels ong>ofong> enzymes involved in hemesynthesis

(Guidotti et al., 2008). It has long been known that

hemaong>toong>poiesis ong>andong> heme synthesis affected by ong>leadong>

poisoning (Doull et al., 1980).

In our study reactive ong>andong> cleaved type ong>ofong>

lymphocyte were observed at the time ong>ofong> birth in

ong>leadong> treated groups which were reinstated by

increased number ong>ofong> plasmacyong>toong>id, reactive, large,

oval, irregular, binucleated ong>andong> cleaved

lymphocytes in further days ong>ofong> lactation. In the

current investigation at higher dose (32 mg) we


found apopong>toong>tic or necrotic neutrophils were more

prominent in the first ong>andong> second week ong>ofong> lactation.

These neutrophils were characterized by 3-4

separate ong>andong> equal lobes with less condensed

chromatin ong>andong> diffuse cyong>toong>plasmic region.

Lead treated group at the termination ong>ofong>

lactation, include abnormal nuclear segmentation,

giving abnormal appearance ong>ofong> nucleus ong>andong>

chromatin condensation in most ong>ofong> the neutrophils

ong>andong> forming a ring like nucleus in some

neutrophils. Villagra et al., (1997) also postulates

that ong>leadong> ong>exposureong> doubles ong>toong>tal ong>andong> segmented

neutrophils in both estrogens treated ong>andong> untreated

rats but causes a three-fold increase in bong>andong>

neutrophils in animals without estrogen treatment,

but not in animals treated with estrogen. With a

disappearance ong>ofong> non-degranulated eosinophils, the

decrease in non-degranulated eosinophils was

under the effect ong>ofong> ong>leadong> ong>exposureong>. He also

demonstrates that prepubertal rat ong>exposureong> ong>toong> ong>leadong>

affects blood neutrophil ong>andong> eosinophil leukocyte

levels ong>andong> induces eosinophil degranulation.

Vyskocil et al., (1991) discovered the effect ong>ofong>

ong>leadong> on bong>andong> neutrophils reveals an increased

neutrophilopoiesis rather than release from

intravascularly sequestered forms in ong>leadong>-exposed

animals.

In ong>leadong> treated group from birth till the

termination ong>ofong> lactation, the number ong>ofong> immature

cells was increased. There was asynchrony ong>ofong>

maturation between nucleus ong>andong> cyong>toong>plasm. During

normal granulocyong>toong>poiesis the lengthening ong>andong>

pinching ong>ofong> the nucleus were coordinated with

progressive condensation ong>ofong> the chromatin with

accelerated maturation nuclear division may be skip

ong>andong> cells retain immature features, because ong>toong>xic

changes ong>ofong> ong>leadong> accompanies a left shift i.e.

presence ong>ofong> immature neutrophils, bong>andong>s,

metamyelocytes, myelocytes ong>andong> other unidentified

immature cells. White Blood Cells generally

increase as compared ong>toong> the control level. The

increase in WBC count indicates the activation ong>ofong>

defense mechanism ong>andong> immune system ong>ofong> gasoline

workers (Whitby, 1980). These findings are also in

confirmations, with our results.

In conclusion, ong>leadong> ong>exposureong> ong>leadong>s ong>toong> various

hemaong>toong>logical disorders in white blood cells

including neutrophilic degeneration, immature

cells, abnormal neutrophils, reactive ong>andong>

plasmacyong>toong>id lymphocyte, reactive monocyte etc.

The present study indicates that after administration

ong>ofong> 266.66, 533.33 ong>andong> 1066.66 mg/kg/body weight

doses ong>ofong> ong>leadong> acetate WBCs show structural

abnormalities in their nucleus ong>andong> cyong>toong>plasm

Prenatal ong>andong> ong>neonatalong> ong>exposureong> ong>toong> ong>leadong> 39

including improper segmentation ong>andong> lesser

condensation ong>ofong> nucleus. Lead causes fluctuations

in the number ong>ofong> various cell types at different

stages ong>ofong> postnatal development. The ong>exposureong> ong>toong>

ong>leadong> possesses the potentials ong>toong> induce hazardous

biological effects during pre ong>andong> postnatal

development in Swiss mice.

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