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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