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IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 1 4 ( 1 & 2 ) , 2 6 1 - 2 8 2 ( 1 9 9 2 )

IMMUNOMODULATORY EFFECTS OF Y -HCH (LINDANE) IN MICE

1 * 2 1 P. Meera , P.R. Rao , R.Shanker and 0. T r i p a t h i *Cell and Molecular Biology Lab. , Department of Zoology, Osmania University, Hyderabad 500 007 'Physiology Division, Central Drug Research Institute, Lucknow 226 001 and 21ndustrial Toxicology Research C e n t r e , Lucknow 226 001, INDIA

ABSTRACT Mice were fed f o r 24 weeks with t h r e e different subtoxic dosages of I - H C H ( 0 . 0 1 2 , 0.12 and 1.2 mglkg) mixed i n powdered feed. The immunological profile was assessed at an interval of one month during t h e entire exposure period. Both t h e cell mediated and humoral components of immunity showed a biphasic response characterized i n i t i a l l y b y stimulation followed b y suppression i n a dose dependent m a n n e r . However, Y -HCH d i d not affect t h e functional properties of peritoneal macrophages. Histological changes i n lymphoid organs were i n accordance with t h e biphasic immunomodulatory effects of Y -HCH.

INTRODUCTION

Y -HCH

(gamma hexachlorocyclohexane, Lindane)

a pesticide

and ectoparasiticide (1) gets undesirable access into t h e human and

animal

food chains, throueh

system skin

continued

absorption,

intestinal

absorption through

placental transfer t o f e t u s ,

m o t h e r ' s milk into the offspring (2,3).

l i p o p h i l i c i t y Y -HCH tissue

via

(4).

As

and

Due to high

gets deposited selectively i n t h e adipose

a consequence to such an accumulation i n the

261 Copyright 0 1992 by Marcel Dekker, Inc.

262

MEERA ET A L .

body, Y -HCH

levels ranging from 0.1 to 1.53 mglkg have been

reported i n various suggested Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by University of Otago on 01/06/15 For personal use only.

and

that

(6-9).

adastic

Some of

anemia

and

reports

causing blood

suppress t h e

have

dyscrasias

immune

status

these hematological effects a r e also suggested

to occur due to 'I-HCH

(9).

Several

is capable of

Lindane

primary

(5).

populations

induced alterations i n t h e immune system

Despite such immunotoxic implications of chronic exposure

to Y -HCH,

t h e time course of changes i n i m m u n e activitv follow-

ing

chronic

yet

known.

administration

of

doses of Y -HCH

subtoxic

I t was investigated

i n present

is not

study bv feeding

mice for 24 wks with different subtoxic doses of Y -HCH

in

powdered

cell

feed

mediated

and

and

by

observing

humoral)

at

the

various

showed a biphasic immunomodulatory

immune

mixed

status

intervals.

(both

The results

effect of Y -HCH.

MATERIALS AND METHODS Young

healthy

Swiss

female

albino

mice

gm were obtained from National Institute of Nutrition,

and

Central

Research

Drug

Institute,

(average body

weight 20 g m )

and

Biology,

Molecular

pellet feed

Chemicals:

Hvderabad

BALBlc

mice

procured from Centre of Cellular

Hyderabad

were

additionallv

used

in

Animals w e r e housed s i x i n each caqe and provided

t h i s study. with

Lucknow.

15-16

weighing

V

(Hindustan Lever ( 9 7 8 ) purity

-HCH

Ltd) and

ad libitum. water -

was obtained

from

Aldrich

Chemical Compnay, USA, growth medium, RPMI 1640 HEPES buffer were serum

purchased

from

GIBCO Laboratories,

(FCS) was from Sera

concavalin A (Con A ) .

Laboratories,

U.K.

and Fetal Calf

U.K.

Mitogens l i k e

Lipopolvsaccharide (LPS) were purchased

from Sigma Chemical Company, USA ,and Mitomycin C from Biochem Pharmaceutical Industries, India. India.

Nutrient

India.

96

broth

round

and

welled

'H-Thymidine

was from BARC,

Agar were obtained from Hi-Media, tissue

culture

plates

were

procured

from N U N C , Denmark.

Plan of -

experiment:

After two

divided into four groups.

weeks

of

adaptation mice were

The oral LD50 value of Y -HCH

in

263

IMMUNE R E S P O N S E TO L I N D A N E

f e m a l e m i c e w a s found to be 120 m g l k g body w e i g h t .

-HCH

w a s m i x e d a s per t h e c o n c e n t r a t i o n r e q u i r e d i n p o w d e r e d f e e d

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a n d e x p o s u r e c o n t i n u e d u p t o 24 w k s as f o l l o w s : Group I G r o u p I1 G r o u p 111 Group IV

Normal 0.0001 0.001 0.01

Control 0.012 m g / k g w t 0.12 mg/kg wt 1.2 mg/kg wt

pellet x LDS0 x LD50 x LD50

S i x a n i m a l s of c o n t r o l a n d Y-HCH exposed m i c e of d i f f e r e n t g r o u p s at the i n t e r v a l of o n e month w e r e e m p l o y e d f o r immunological tests.

S t u d i e s on h i s t o l o g y of t h y m u s , peripheral l y m p h

n o d e s a n d s p l e e n w e r e carried o u t at 4 , treatment.

The organs

were fixed in

12 a n d 24 w k s post 10% b u f f e r e d formalin,

dehydrated i n g r a d e d e t h a n o l a n d e m b e d d e d i n p a r a f f i n . S e c t i o n s of

5

um

thickness

techniques.

were

prepared

following

Multiple sections w e r e then

the

conventional

s t a i n e d w i t h hemato-

x y l i n a n d eosin (10).

Prepamtion of Spleen cells: Isolated s p l e e n cells w e r e o b t a i n e d by g e n t l y

RPMI RPMI

p r e s s i n g the s p l e e n on the f i n e steel m e s h i n cold

1640.

T h e cells o b t a i n e d

1640 s u p p l e m e n t e d

mM HEPES b u f f e r ,

with

w e r e w a s h e d thrice i n c o l d

10% FCS,

2 mM L-glutamine,

20

100 u n i t s / m l p e n c i l l i n a n d 100 u g / m l S t r e p t o -

m y c i n at 600 g i n a c o l d c e n t r i f u g e .

T h e cell viability w a s

checked by t r y p a n b l u e e x c l u s i o n t e c h n i q u e a n d cell p o p u l a t i o n

w a s a d j u s t e d a f t e r counting t h e cells in N e u b a r h a e m o c y t o m e t e r

Delayed Type Hypersensitivity Reaction (DTH) :

.

T h e DTH r e s p o n s e

t o SRBC i n m i c e w a s s t u d i e d by the m o d i f i e d m e t h o d of Langrange

et a1 (11).

B r i e f l y , the a n i m a l s w e r e s e n s i t i z e d w i t h a n i n t r a -

5% ( v l v ) SRBC s u s p e n s i o n i n sterile s a l i n e i n t o the r i g h t f r o n t f o o t pad. On day 5 a c h a l l e n q -

dermal

injection

i n g dose

of

0.02

ml

of

of 0.02 m l of 5% ( v / v ) s u s p e n s i o n of SRBC i n sterile

s a l i n e w a s s i m i l a r l y i n j e c t e d i n t h e r i g h t h i n d f o o t pad.

The

i n d u r a t i o n w a s read 24 h r s later u s i n g v e r n i e r callipers. B e s i d e s , the f o o t pad s k i n w a s also e x a m i n e d f o r h i s t o l o g i c a l c h a n g e s .

264

MEERA ET AL.

&mDhocvte and

Transformation:

Knight

studies.

The

(12)

was

followed

Single

cell

suspension

modified for of

method

lymphocyte

of

Thrope

transformation

splenocytes

from

control

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and treated mice were prepared a s described above. cells were incubated

i n 96 well round- bottom microtitre

w i t h 2x105 cellslwell i n 0.1 m l .

concentration and

of

Spleen

,

mitogens

LPS at 10 pglwell.

plate

After determining the optimum

Con

A

was used

at

1.25 pglwell

A l l cultures were r u n i n triplicates

and t h e culture plates incubated a t 37OC i n GO2 incubator (5% 72 h r s ) .

C02,

pulsed

with

mCilmM).

After 48 h r s of

0.5

3H-thymidine Subsequently

the

automated cell harvester

incubation the cultures were

pCilwell plates

(specific activity

were

harvested

(PHD Cambridge Technology,

washed with 0.9% saline, followed by 10% TCA.

using

600 an

USA) and

The precipitates

thus obtained were washed with methanol, d r i e d overnight and transferred into scintillaltion vials.

The cocktail used consisted

of 5.5 gm of PPO with 500 mg of POPOP i n one litre of toluene.

Incorporated (CPM) i n

radioactivity

Packard

Liquid

was

counted

as

Scintillation

counts

per m i n u t e

counter.

The

results

were expressed a s stimulation index ( S . I,) obtained by equation (1). S.I. =

......( 1)

Average cprn of mitogen containing culture Average cpm of control culture

Mixed Lymphocyte Reactions (MLR):

The method followed for

the one way proliferative response of lymphocyte to alloantigen was

essentially t h e

(13). mouse

The

same

as

described by

splenocytes collected

represented

the

stimulator

from the cells

Bach

and

Voynow

spleen

of

BALBlc

while

splenocytes were derived from control o r I -HCH albino mice.

the

responder

treated S w i s s

The stimulator cells were initially treated with

mitomycin C (25 pglml) and incubated for 30 m i n i n C02 incubator (37OC,

5% C02).

Following incubation the cells w e r e washed

thrice and dispensed i n RPMI 1640 supplemented with 10% FCS. The stimulator

cells (0.1

m l containing lx104 cells/well were

265

IMMUNE RESPONSE TO LINDANE

incubated

responder

splenocytes

( 0 .I

m l containing

2x105

cellslwell) i n 96 well round bottom plates.

For the background

control

and

mice Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by University of Otago on 01/06/15 For personal use only.

with

the

were

splenocytes incubated

from

with

the

control

mitomycin

C

in

Y -HCH

the

treated

same manner.

A l l the cultures were r u n i n triplicates and incubated i n C02 incubator ( 5 days, thymidine,

5% C02).

harvested

The cells were pulsed with 3H-

and counted as described

previously

for

The results were expressed a s stimulation index

radioactivity.

determined by equation( 2 ) Average cpm of BALBlc stimulated cultures ...(2 ) Average cpm of S w i s s albino stimulated cultures

S.I.=

Haemolytic Plaque Forming Cell Assay:

(i$)

of IgM antibody

method was followed for the enumeration

Both T-dependent antigen (SRBC)

forming cells i n the spleen. and

Cunningham and Szenburg's

T-independent

antigen

(LPS) were used.

SRBC

Coating of

with LPS was carried out according to the method of Halliday and Webb

To 1 m l of

(15).

phosphate buffered saline,

0.5

m l of packed SRBC and 0.1 mg of LPS were added and incubated for 1 h r at 37OC.

Following incubation the cells were washed

thrice and 10% ( v l v ) suspension made i n served as T-independent antigen.

sterile saline.

This

0.2 m l of T-dependent (SRBC)

and T-independent antigen (LPS coated SRBC) was injected intraperitoneally to separate group of

mice on day zero.

5 the

single cell

spleen

was

removed

and

On day

suspensions made

a s described earlier. 0.15 m l of cell suspension containing 5 5x10 cells, 0.02 m l of 20% SRBC or LPS coated SRBC and 0.03 ml

of

complement

(g.pig

serum) was taken

in

a tube,

mixed

and then dispensed i n the form of monolayer i n Cunningham's chamber

(a slide covered by the coverslip over an adhesive

tape to form a gap and sealed).

This monolayer suspension

of the assay mixture was incubated for 45 minutes at 37OC and counted

for the

plaques

under low power of

The results are expressed a s number of 6 10 lymphocytes.

the microscope.

plaque

forming cell/

MEERA ET A L .

266

Macrophage

Phagocytic

of peritoneal

Activity:

The

bactericidal

activity

macrophages was studied b y the method of

Van

Furth et a1 ( 1 6 ) with slight modification. Peritoneal macrophages

m l of 0.2

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were activated by injecting 0.1 peritoneally were

two

lavaged

days

out

m n l m l of LPS intra-

before

the

experiment.

cold

RPMI

1640 from

with

Macrophages the

peritoneum

of the mouse on the t h i r d day after the injection of LPS. Macro-

phages

and

Staphylococcus

aureus

the

the

assay

agar.

mixture

was diluted

The number of

subtracting

the

number

of

1:lOO

in

ratio

After lysing with 1 m l of s t e r i l e

and incubated for 30 min. water

mixed

were

bacteria

serially and plated killed

surviving

on

w e r e obtained

bacteria

by

represented

by

the colony counts from t h e number of bacteria added initially.

The results w e r e expressed a s percent bactericidal activity. RESULTS

Histological changes

Lymphoid Orgaus:

Thymus a t 4 wks of Y-HCH

treatment revealed

an increase i n t h e size of medulla with corresponding decrease in

cellular

of

population

cortex

There was no manifestation of -HCH feeding.

which

was

dose dependent.

cytotoxicity upto t h i s state of

A t 1 2 w k s it showed decreased cortical lympho-

A few necrosed c e l l s could also be seen predominantly

cytes.

i n t h e medulla together with congestion of blood vessels. However at 24 wks t h e r e was severe loss of cortex and medulla.

distinction

between

the

The thymic lobules demonstrated marked

decrease i n t h e cortical lymphocytes and a prominent medulla with many necrosed cells (Fig. 1 and 2). Lymph node demonstrated increased activity i n t h e lymphoid follicles

together

with

prominent

mature plasma cells a t 4 wks.

medullary At

cords

containing

12 wks of treatment

the

changes i n lymph node resembled closely to t h a t seen a t control. However, at 24 w k s t h e demarcation between cortex a n d paracortex

was

lost

accompanied

by

considerable dep.ession i n -1

267

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IMMUNE RESPONSE TO LINDANE

Fig. 1: Section of thymus of mouse a t 12 weeks post treatment with V -HCH (1.2 mg) showing presence of necrosed cells (9) i n medulla. Hematoxylin and eosin, x510.

ocyte

population

throughout

the

node.

Marked

reduction

in

s i z e of medullary cords was also noted (Fig. 3 and 4 ) . Spleen wks of

of

did

not

treatment

megakaryocytes.

reveal

except

any

that

there

The lymphoid

at 12 wks post treatment.

significant was

reaction

active

after

4

proliferation

follicles appeared reduced

Finally 24 wks of treatment resulted

i n considerable reduction i n the overall cellularity of red pulp and white pulp areas (Fig. 5 ) . Foot

pad

exhibited

a

dose

dependent

increase

followed

by decrease i n the cellularity of mononuclear cells i n dermis. These changes corresponded to the two phases of immunomodulation of J-HCH

(Fig. 6 and 7 ) .

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268

MEERA ET AL.

Fig. 2:

Thymus of mouse at 24 weeks post treatment with Y-HCH ( 1 . 2 mg) containing many necrosed cells i n medulla. H%E, x510.

-.Immunological changes

Delayed Type Hypersensitivity Reaction: f o r DTH reaction i n mice following I - H C H in Table 1. of

Measured i n terms of

mouse foot pad,

dose-dependent by inhibition

results obtained

exposure a r e presented

t h e diameter of

induration

DTH response showed a time-dependent and

change

with

upto 24 wks.

at 4 wks (P

Immunomodulatory effects of gamma-HCH (Lindane) in mice.

Mice were fed for 24 weeks with three different subtoxic dosages of gamma-HCH (0.012, 0.12 and 1.2 mg/kg) mixed in powdered feed. The immunological pr...
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