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4. EXPERIMENTS
CHAPTER 4.1
CHANGES IN ANTIOXIDANT ENZYMES AND ANTIOXIDANTS IN MASTOMYS
NATALENSIS AFTER BRUGIA MALAY1 INFECTION AND ITS RELATION TO
DEC TREATMENT
4.1.1. MATERIALS AND METHODS
Male animals of the age of 6-8 weeks, weighing 35-40 gms were used for the study.
A group of 20 animals was inoculated subcutaneously with 200 infective larvae of B. malayi
subpenodic strain, while another group of 20 animals was kept as control. The animals were
maintained for a maximum period of 120 days.
Four animals from each group were necropsied on 0, 30, 60,90 and 120 days post
inoculation and assayed for antioxidants and antioxidant enzymes as per the methods given
below. Organs such as liver, testes, bwin, heart and lungs were excised out, washed
thoroughly with saline and blotted dry. A 10% (wlv) homogenate was prepared in Tris-HCI
buffer (0.01 M, pH 7.4) containing 0.25 M sucrose using a potter elvehjem homogeniser
fined with a power driven teflon pestle. For assays of mitochondnal and cytosolic enzymes,
subcellular fractionation was done by centrifugmg the 10Ph homogenate in a Sorval (RC5C)
refrigerated (4'C) centrifuge. The nuclear fiaction was obtained by centrifuging at 800g for
ten minutes and the mitochondrial fiaction was obtained by centrifuging at 8000g for 20
minutes. The post mitochondria1 supernatant was centrifuged at 100,000g (Sorval-OTD-50)
for I hr to isolate the mimsomes. The soluble supmatant served as cytosol.
4.1.1.1 Superoxide dismutase
Superoxide dismutase is estimated by the method of Marklund and Marklund (1974).
' h e dtgnt of inhibition of autoxidation of pyrogallol at an alkaline pH by SOD was
pa a measwe of the enzyme activity.
Ragenb 1. Tris-HCI buffer . : 0.1 M, pH 8.2 containing 2 mM of diethylene hiamine penta
acetic acid.
2. Tris- HCI : 0.05 M, pH 7.4.
3. Pyrogallol stock solution : 25.2 rng of pyrogallol was dissolved in 1 ml of 0.05 M Tris-
HCI buffer, pH 7.4 in a test tube stoppered and wrapped with an aluminum foil.
4. Pyrogallol working solution : At the time of assay 0.5 ml was diluted to 50 ml with 0.05
M TricHCl buffer, pH 7.4 to give a 2 mM solution and shielded from exposure to light.
5. Absolute ethanol.
6. Chlomfonn.
Partially purified SOD was prepared as described by McCord and Fridovich (1969).
To 1 ml of the tissue homogenate, 0.25 ml of absolute ethanol and 0.15 ml of chloroform
was added. After 15 minutes of shalung in a mechmcal shaker, the suspension was
centrifuged and the supematant obtained constituted the enzyme extract. The reaction
mixture for autoxidation consisted of 2 ml of the buffer containing DETAPAC, 0.5 ml of 2
rnM pyrogalbl and 1.5 ml water. Irutially, the rate of autoxidation of pyrogalbl was noted at
an interval of one minute to three minutes. The assay mixture for the enzyme contained 2 ml
of 0.05 M Tris-HCI buffer, 0.5 ml pyrogallol, aliquots of the homogenate and water to give a
final volume of 4 ml. The rate of inhibition of pyrogallol autoxidation after the addition of
the enzyme was noted. Iron accelerates pyrogallol autoxidation even in trace amounts.
DETAPAC acts as a chelator and thus prevents the interference from ~ e ~ ' as well as from
CU" and hIn2*.
The enzyme activity was expressed in terms of unitsimg protein in which one unit
c o r n & to the amount of enzyme that mhibited the autoxidation reaction by 50%.
The activity of catalase was assayed by the method of S~nha (1972).
The caFakPc m y m e pnparation was allowed to split hydrogen peroxide for
~ifFcmnt paiod of time. The reaction was stopped at specifibdgme intervals by adding
dichromate/acetic acid mixture. The dichromate in acetic acid is reduced to chromic acetate,
whcn heated in the presence of hydrogen peroxide with the formation of perchloric acid as
an unstable intermediate. The chromic acetate thus produced is measured at 610 nm. Since
dichrornate has no absorbance in this region, the presence of this compound in the assay
mixture does not interfere with the calorimetric determination of chromic acetate.
Resgents
I. Stock dichromatdacetic acid reagent : This reagent was prepared by mixing a 5%
solution of potassium dichromate with glacial acetic acid (1 :3) by volume.
2. Working dichromatdacetic acid reagent : The stock was diluted (1:s) with water to
make the working dichromatdacetic acid solution.
3. Hydrogen peroxide (0.2 M) : 1.0 ml of 30% hydrogen peroxide was made
upto 45.0 ml with water.
4. Phosphate buffer : 0.01 M, pH 7.0
The reaction mixture contained 0.5 ml of hydrogen peroxide, 1.0 rnl of buffer, 0.4 ml
of water and 0.1 ml of diluted homogenate (1: 10). After 15, 30 and 60 seconds of incubation,
2.0 ml of dichromateJacetic acid reagent was added. To the control tube, the enzyme was
added after the addition of acid reagent The tubes were then heated and the colour
developed was read at 610 nm.The activity of catalase was amved at from the amount of
hydrogen peroxide consumed and was expressed as p o l e s of hydrogen peroxide
consumed/rninutdmg protein.
'Ibis was assayed by the method of Beutler (1984).
GST catalyses the reaction of I-chloro, 2,4-dinitrobenzene with the -SH group of
glutahone. The conjugate formed is measured at 340 nrn.
Reagents
1. Potassium phosphate buffer : 0.5 M, pH 6.5
2. I-chlm -2,4 dinitrobemne (CDNB) : 30 mM in 95% ethandl
3. Reduced glutathione : 30 mM
To I ml of buffer and 0.1 ml of CDNB, 1.7 ml of water was added and incubated at
37'C for 5 minutes. After incubation, 0.1 ml of reduced glutathione was added and fwther
incubated for 5 minutes. The reaction was initiated by addmg 0.1 ml of the homogenate.
The increase in absorbance was measured at 340 nrn at one minute interval for 5
minutes. The values are expressed as unitslmg protein.
4.1.1.4 Glutathione Peroxidase
Glutathione peroxidase was assayed by the method of Rotruck et al. (1973) with
some modifications.
Ln this procedure, the rate of oxidation of glutathione by Hz& is used as a measure of
peroxidase activity. Glutathione remaining in the solution at a given time is determined by its
reaction with DTNB. EDTA is used in the incubation medium to reduce the non-enzymatic
reaction rate at a low level.
Reagents
1. Sodium phosphate buffer : 0.3 M, pH 7.0
2. Sodium azide : 1Omhl
3. Reduced glutathione : 4 m M
4. Hydrogen peroxide :2.5 mM
5. TCA : lPh
6. Phosphate solution : 0.3 M disodium hydrogen phosphate.
7. DTNB : 40 mg/100 ml of 1% sod~um cihate
8. EDTA : 0.8 mM
9. Standard : 20 mg of reduced glutathione in 100 rnl distilled water.
This solution contained 20 pg of glutathione/O. 1 ml.
A known volume of the homogenate was added to the incubation medium which
containal0.4 ml of buffer, 0.2 rnl of sodium azide, 0.2'ml of ED%, 0.2 ml of Hydrogen
paoxide and 0.2 ml of reduced glutathione. The incubation medium was made upto a final
volume of 2.0 ml with water. The tubes were incubated at 37'C for 90 and 180 minutes. The
d o n was terminated by the addition of 1.0 ml of the precipitating agent. The reaction
mixture was centrifuged and to the supernatant, 6.0 ml of disodium hydrogen phosphate was
added. One ml of DTNB reagent was added just prior to the calorimetric analysis. The
absorbance was read at 412 nm against a blank, which contained only 6.0 rnl of disodium
phosphate and 1.0 ml of DTNB reagent. Suitable aliquot5 of the standards were taken and
treated in a similar manner. The activity was expressed in terms of pgm of glutathione
utilizedlrninutelmg protein.
4.1.1.5 Glutathione reductase
Glutathione reductase was assayed according to the method of Beutler (1984).
Glutahone reductase catalyses the reduction of o x i W glutathione (GSSG) by
NADPH or NADH to reduced glutathione. The activity of the enzyme is measured at 340
mn following the oxidation of NADPH. GR is a flavin enzyme and it has been found that it
is not fully activated by FAD in normal samples. Complete activation of apoenzyme requires
the preincubation of enzyme with FAD. This is done prior to the addition of GSSG or
NADF'H to the reaction system.
Reagents
1. Tris-HC1 : 1 M with EDTA 5 mM, pH 8.0
2. FAD : l o p
3. Oxidised glutathione (GSSG) : 0.033 M
4. NADPH :2mM
To 100 p1 of Tris-HCI buffer, 20 tr] of the sample, 1.58 ml of water and 200 pl of
FAD is added and incubated at 3PC for 10 minutes. To the test sample, 200 tr] of GSSG is
lddcd and again incubated at 3PC for 10 minutes. Later 100 tr] of NADPH is added to both
the blank and the test samples and the decrease in optical density was measured against the
at 340 tun (3PC). The values are expressad as uniWgm p r o t h
4.1.1.6 Glucose-6-phosphate dehydrogenase
This enzyme was assayed by the method of Z i et a/. (1958) with some
modifications.
Glucose-&phosphate dehydrogenase is assayed by measuring the increase in
absorbance, which occurred at 340 nm when NADP is reduced to NADPH. The reduction
takes place when two electrons are transferred fkom glucose-6-phosphate to NADP in the
reaction catalysed by glucose-&phosphate dehydrogenase.
Reagents
I. NADP : 0.2 mM
2. Tris-HCI buffer : 0. l M, pH 8.0
3. Magnesium chloride : 0.1 M
4. Glucose-6-phosphate : 6 mM
To 0.1 ml of NADP, buffer and MgC12, 0.5 ml of water and 0.1 ml of the
homogenate was added. After 10 minutes, the reaction was initiated by the addition of 0.1 ml
of glucose-6-phosphate solution. The increase in optical density was measured at 340 nm at
2S°C. The activity of the enzyme was expressed in terms of units/mg/protein where one unit
cornponds to the amount of the enzyme required to bring about a change in optical density
of 0.01 /minute.
4.1.1.7 Reduced glutathione
Reduced glutathione was estimated by the method of Moron eta/. (1979)
This method is based on the development of yellow colour when 5,5'dithio-bis-2-
n ~ t r o b i c acid (DTNB) is added to the compounds containing sulphydryl p u p s .
Reagent6
1. DTNB : 0.6 mM in 0.2 M phosphate buffer, pH 8.0.
2. Phosphate buffer : 0.2 M, pH 8.0.
3. TCA : 5%
4. Standard : 10 mg of reduced glutathione in I00 ml water. This contained
10pgm of glutathione in 0. I ml solution.
To 0.1 mi of the homogenate, 1.0 ml of water and 2.0 ml of precipitating agent were
added, mixed thoroughly and centrifuged after 5 minutes. To an aliquot of the supernatant,
2.0 ml of DTNB reagent was added and made upto a final volume of 3.0 ml with phosphate.
buffer. The absorbance was read at 412 nm against a reagent blank The amount of
glutathione was exprrssed as pgm of glutathionel mg protein.
4.1.1.8 Total thiol groups
Total thlol groups were estimated acconhg to the method of Sedlack and Lindsay
(1 %8).
The sulphydryl p u p s in tissues were determined by using the Ellman's reagent. In
this method, DTNB is reduced by -SH groups to form 1 mole of 2-nitro, 5-mercaptobenzoic
acid per mole of -SH.
Reagents
1.0.01 MDTNEI : 99 mg DTNEI dissolved in 25 ml of absolute methanol.
2. Tris-HCl buffer : 0.2 M, pH 8.2 containing 0.02 M EDTA.
The sampk was pnpartd by homogenising 100 mg of tissue in 4.0 ml of 0.02 M
EDTA. To an aliquot of the tissue homogenate, 1.5 ml of Tris-HCI buffer @H 8.2) and 0.1
ml of DTNB was added, mixed and made upto 10.0 ml with absolute methanol. A reagent
blank without the sample and sample blank without DTNEI were prepared in the same
manna. The test tubes were stoppered and allowed to stand with occasional h k m g for 15
minutes. The reaction mixture was cenhifuged at 3000g at R.* for 15 minutes. The
absorbance of the clear supernatant was read at 412 nm. Calibration curves were obtained
with reduced glutathione as standard. Values were expressed as pgm of glutathiondmg
protein.
4.1.1.9 Ascorbic acid
Ascorbic acid was estimated by the method of Omaye ef al. (1979).
Ascorbic acid is oxidzed by copper to form &hydroascorbic acid and
diketogluconic acid. When treated with 2.4, dmirophenyl hydrazie, it reacts to form the
derivative bis 2,4, dinitrophenyl hydrame. This compound in strong sulphuric acid
undergoes a rearrangement to form a product with an absorption band that is measured at
520 nm. The reaction is run in the presence of thlourea to provide a mildly reducing medium,
which helps to prevent the ~nterference from non-ascorbic chromogens.
Reagents
1. TCA : 5%
2. DTC reagent : 3 gms DNPH (ditropbenyl hydrazine), 0.4 gms thiourea and 0.05 gms of
copper sulphate were dissolved in 10 ml of 9N sulphuric acid and made upto 100 ml with the
same solution.
An aliquot of the homogate was precipitated with ice-cold TCA, cenhihged for 20
minutes at 3,500g and 1.0 ml of the supernatant was made upto 3.0 ml with 5% TCA and
then, treated with 0.2 ml of DTC and incubated for 3 hrs at 37'C. Then 1.5 ml of icecold
65% sulphuric acid was added, mixed and kept at RT. for an additional 30 minutes. Blank
contained only 3.0 ml of TCA. Standards in the range of 10-50pgms were treated in the
same way. The intensity of the color was measured at 520 nm. Results were expressed as
pgm of ascorbic acid! mg protein.
4.1.1.10 Lipid peroxidation
Lipid peroxidation in the tissue was estimated by the method of Stoch and
Dormandy (197 1 ).
Malondialdehyde (MDA), produced during peroxidation of lipids, served as an index
of lipid pemxidation. In this method MDA reacts with thiobarbihuic acid to generate a
coloured product, which is read at 532 nm.
Reagents
I. Isotonic phosphate buffered saline (PBS), pH 7.4.
2. TBA : 1% in 0.05 m M i t sodium hydroxide.
3. TCA : 10°/o solution in 0.1 moVlit d m n arsenite.
4. Standard MDA : stock solution of MDA (400 nmol/ml) was prepared using 1,1,3,3
tetraethoxy propane. This was stored at 4OC and diluted with distilled water to make
working standard of 50 d m 1 concentration.
5. EDTA : 0.1 momit.
One rnl of the homogenate was suspended in 0.1 ml of PBS and 0.5 ml of TCA,
followed by 2.0 ml TBA and 0.075 ml EDTA. Tubes were mixed and kept in a boiling water
bath for I5 minutes. Tubes were cooled at R.T. and centrifuged. Absorbance was read at 532
nm. Each test sample had its own blank tube, which was not boiled. Subtraction of
absorbance of unboiled sample from boiled sample eliminated absorbance increase due to
any non-TBA reactive material in the sample. EDTA was added to chelate any ironlother
metal in the ahac t which, otherwise can initiate lipid peroxidation during boiling and may
result in falsely elevated TBA reactivity. Results were expressed as nmoles of MDA
fonndmg protein! 20 minutes.
4.1.1.11 Protein
Protein was estimated by the method of Lowry et al. (I%])
-eats
I . Lowry's reagent :Solution A: 2% sodium carbonate in 0.1 N sod~um hydroxide solution.
: Solution B: 0.5% copper sulphate in 1% sodium potassium tartarate.
The reagent was prepared by mixing 50 ml of solution A with 1.0 ml of solution B
prior to use.
2. Folins Ciocalteau w e n t
To 0.1 ml of (1:lO) diluted homogenate, 0.9 ml of water and 5.0 ml of Lowry's
reagent was added, mixed well and kept at RT. for 10 minutes. To this, 0.5 ml of Folin's
reagent was added and mixed.
Standard Bovine serum albumin solution containing 20-100 micrograms of protein
and a blank were treated in a similar manner. The intensity of the colour developed was
measured after 20 minutes at 660 nrn. The values were expressed as m g / p tissue.
4.1.2 RESULTS
The experimental animals w m necropsied on 0 day and on 30,60,90 and 120 days
post-inoculation with infective larvae of B. malayi for biochemical studies. On 120th day
post-inoculation, various organs were examined for the presence of adult worms. The worm
recovery was 25.1 5linfected animal and they were distributed in lungs, testes and heart and
liver and brain did not have adult worms. Lungs contained the maximum number of worms
followed by testes and heart (F1g.2).
Biochemical analyses were done to determine whether the B.malayi infection caused
any change in the production of oxygen €ree radicals, antioxidants and antioxidant enzymes
in different organs of the host animal, M. natalemis.
Fig 2:B.malayi distribution in Lungs testes & heart of M-natalensis
Lungs (44.38)
Testes (37.07)
Figures in parenthesis a r e %age of adult
worms recovered
4.1.2.1 Changes in antioxidant enzymes and antioxidants in control and
B.m&yi infected animals
4.1.2.1.1 Superoxide dismutase
The changes in the activity of SOD in the organs of infected and control animals are
presented in Figs. 3-5. In control animals, all the organs (viz., the liver, testes, brain, heart
and lungs) showed a continuous increase in the activity of this enzyme from 0 to 120 days.
Contrary to this, in the testes, heart and brain of the infected animals, the activity of this
enzyme showed an increasing trend upto 30 days and thereafter the enzyme activity declined
s~gn~ficantly w0.05), while in the liver, the activity increased sigmficantly upto 60 days and
thereafkr declined. And in the lungs the enzyme activity showed increasing trend throughout
and the activity was higher than that of control .
4.1.2.1.2 Catalase
The changes in the catalase activity are shown in Figs. 6-8. All the organs of the
control animals showed increase in catalase activity from 0 to 120 days. Compared to this,
the tesw, heart and brain of the infected animals showed decline in the enzyme activity from
30th day while liver showed significant m . 0 5 ) decline from 60th day. However, the lungs
showed a steady increase in catalase activity from 30 days (significant at F0.05) and the
increase was much higher than that observed in control.
4.1.2.13 Clutathione-s-transferase
The activity of GST in various organs of the infected and control animals are shown
in F~gs. 9-1 1. The GST activity was found to increase in control animals from day 0 to
day 120.
LIST OF ABBREVIATIONS USED IN THE GRAPHS
Liver - Li
Testes - T
Heart - H
Brain - B
L u g s - Lu Normal - N
Infected - 1
Na' K' ATPase - Na
ca2' ATPase - Ca
Mg 2' ATPase - Mg
Haemoglobin - Hb
Acetylcholinesterase - Ach est
y-glutarnyl transpeptidase - G P
Alkaline phosphatase - AP
C a t a l e -CAT
Fig.3: Effect of B.malayi on SOD in in Liver and Testes of M.natalensis
0 30 w so 320
Days of lnfecuon
LI-N + LI-l - T-N - T-1
Fig.4: Effect of B.malayi on SOD in Brain and Heart of M.natalensis
4 -
0 30 60 90 120
Days of Infection
+ B-N --t 8-1 - H-N e H-l
Fig.5: Effect of B.malayi on Lungs of M. natalensis
30-
25 -
0 30 W 90 120
Days of Infection
Fig.6: Effect of B.malayi on Catalase in Liver and Testis of M.natalensis
- 9 16 ; 5 :iq 12
10
e 30 BO UO i 2 0
Days of Infection
-- t i - N + La-l - T-N * T-1
Fig.7: Effect of B.malayi on Catalase in Brain and Heart of M.natalensis
0 30 60 90 120
Days of Infection
c B-N -+- 6-1 - H-N - H-l
Fig.8:Effect of B.malayi on Catalase in Lungs of M. natalensis
"- 34 -
1 32- -4
3 - 9 30-
= 21). 8 I 20
24 - 22
30 60 90 120
Days of Infection
Fig.9: Effect of B.rnalayi on GST in Liver and Testis of h4.natalensrs
2 4 . . 2 2-
1 -
Days of lntect~on
+ b-N -+- LI-1 - T-N -++ T-l
Fig.10: Effect of B.malayi on GST in Brain and Heart of M.natalensis
= 0.
0 2
I 0 30 80 SO 120
Days of Infection
t 8.N --c 6-1 - H-N - H-1
Fig.11: Effect of B.malayi on GST in Lungs of M.natalensis
0 0.
0 55-
Days ot \ntection
Whereas in the infected animals, organs such as testes, brain and heart showed significant
0 . 0 5 ) decline in GST activity from 60 to 120th day while the liver showed a decreasing
bend from 90 to 120 days. However, in the lungs, the enzyme activity showed a significantly
wO.05) increasing bmd from60 to 120 days, but was lower than that of the control.
4.1.2.1.4 Glutathione peroxidase
The organs such as testes, brain and heart of the infected animals showed an increase
in GPx activity upto 30 days (Figs. 12-14) and thereafter it declined gradually to significant
(pcO.05) level as against an increase in the c o m l s . The liver showed significant decline
w . 0 5 ) from 0 to120 days while the lungs showed significant increase w0.05) from 30
days as in the case of control. However, the increase in enzyme activity in infected lungs was
much lower than that observed in the control
4.1.2.1 .S Glutathione reductase
The activity of GR in Liver, testes, brain and heart of the mfected animals was found
to decrease significantly @<0.05) from 0 to 120 days as against an increase in the controls
(Figs. 15-17). In lungs, the activity although decreased from 0 to 30 day, it showed an
increasing trend (significant at ~ 0 . 0 5 ) thereafter as in control, but was lower than that of the
control values.
4.1.2.1.6 Glucose 6 phosphate dehydrogenase (G6PDH)
G6PDH activity increased from 0 to 120 days in all the organs of the conml animals
(Figs. 18-20). In the liver, tests and heart of infected animals, its activity decreased
sigruficantly w . 0 5 ) frwn 30 to 120 days and in the brain from 0 to I20 days. However, in
the lungs its activity increased significantly (pcO.05) from 0 to 120 days as in controls, but
was lcssa than that of the control.
Fig.12: Effect of 6.rnalayi on GPx in Liver and Testis of M.natalensis
22.
21- .
14-
13-
12, '. 30 W 9 0 120
Days of Infection
-) LI-N C LI-l - T-N - T-l
Fig.13: Effect of 6.malayi on GPx in Brain and Heart of M.natalensis
111,
10- .
g 14.
3 # - 2 12-
= 10- - t% El z 8-
11-
4. 0 3 0 M 90 120
Days of lntoct~on
Fig.14: Effect of B.malayi on GPx in Lungs of M. natalensis
10-
0 s-
0 30 80 9 0 120
Days of Infection
Fig.15: Effect of B.malayi on GR in Liver and testes of M. natalensis
0 30 M) 80 120
Days of Infection
Fig. 16: Effect of B.malayi on GR in Brain and Heart of M.natalensis
a - g 25-
to-
0 30 60 90 120
Days of infection
+ 6.N --C B-I - H-N - H-l
Fig.17: Effect of 6.malayi on GR in Lungs of M.natalensis
0 30 90 120
Days of infection
Fig. 18: Effect of B.malayi on G6PDH in Liver and Testes of M. natalensis
4 - 3 5-
3 -I I 30 M 90 120
Days of Infection
-) LI-N 4 b-l - T-N - T-l
Fig.19: Effect of B.malayi on G6PDH in Brain and Heart of M.natalensis
5 i 30 M So 110
Days of Infection
-, 8.N - 8.1 - H-N - H-l
Days of Infection
Lu-N -+- Lu-l
Fig.20: Effect of B.malayi on GGPDH in Lungs of M. natalensis
16-
1s.
g 14.
/
% 13, /
i ./," 1
120
4.1.2.1.7 Reduced glutathione
The GSH levels in the control animals showed an increasing trend throughout the
study period (Figs. 21-23). Whereas, in the infected animals, the liver, testes and heat
showed significant w0.05) decrease in the activity from day 30 to day 120 and in the brain
from day 0 to day 120. However, the lungs showed increasing trend from 30 days to 120
days as in control, but the activity was lower than that of the control.
4.1.2.1.8 Total thiol group
The total hi01 status in the organs of the control animals showed a steady increase
from 0 to 120 days (Figs. 24-26), whereas, the liver, testes and brain of the infected animals
showed a significant decrease w . 0 5 ) . The heart also showed similar trend, but from 30th
to 120thday only. In the lungs, however, the thiol level declined from 0 to 30 days and then
increased significantly wO.05) as in the control. But the values were lower than the control.
4.1.2.1.9 Ascorbic acid
Ascorbic acid content in the organs of mfected and contml animals are p m t e d in
Figs. 27-29. In the organs of the control animals it increased from 0 to 120 days whereas, in
the infected animals, significant decrease w0.05) was observed throughout the study pencd
in testes and heart and it decreased in liver and brain from 30th day to 120th day. However,
in the lungs significant increase ( ~ ~ 0 . 0 5 ) was noticed from 30th day to 120th day and the
values were lower than that observed in control.
4.1.23 Lipid peroxidation
The level of lipid peroxidation was measured in terms of malondialdehyde
released and is presented in Figs. 30-32. The LPO was significantly higher w0.05) in
liver, testes, brain and heart of both control and B.m~layc infected animals. On the
contrary, in the lungs of infected animals there was significant wO.05) reduction in LPO
levels as compartd to the controls.
Fig.21: Effect of B.malayi on GSH in Liver and Testis of M.natalensis
12-
30 w 120
Days of tnfectlon
Fig.22: Effect of B.malayi on GSH in Brain and Heart of M.natalensis
+ 8-N + 8-1 -C H-N ---+ H-1
3 m- 15-
lor,
Fig.23: Effect of B.malayi on GSH in Lungs of M.natalensis
7 -.
Days of infection
54 0 30 0 90 120
Days of infection
+ LI-N + b-l - T-N C- T-1
Fig.24: Effect of B.malayi on SH Group in Liver and Testis of Mnatalensis
Fig.25: Effect of B.rnalayi on SH Group in Brain and Heart of M.natalensis
Days of infection
. -
Fig.26: Effect of B.malayi on SH Group in Lungs of M.natalensis
6-
4- 0 3 0 W 00 120
Days of ~nfecbon
5 12-
g 10- =
Days of infection
L,,.N . . Lu-l
sf!
Fig.27: Effect of B.malayi on Ascorbic acid in Liver & testes of M.natalensis
2 0 ~ I
64 1 30 00 90 1 zc
Days of Infecbon
Fig.28: Effect of 6.malayi on Ascorbic acid in Brain and Heart of M.natalensis
301 I
04 I 30 6a 80 1 2 C
Days of infection
B-N - 0.1 --- W.N + n.1
Fig.29: Effect of B.malayi on Ascorbic acid in Lungs of M.natalensis
9 57
0 -
Days of inteetlon
Fig.30: Effect of B.malayi on LPO in Liver and Testis of Mmatalensis
Days of infection
Fig.31: Effect of B.malayi on LPO in Brain and Heart of M.natalensis
137
12-
1 1 -
Days of lntecbon
Fig.32: Effect of B.malayi on LPO in Lungs of M.natalensis
a- ? 5 .
s 6,"
-9 6 .
5 5 8 -
= ,- 4 5.
44 0 90 120
Days of cnfsction
4.1.2.3 Change in the antioxidant enzymes, antioxidants and lipid
peroxidation in infected animals after Diethylcarbamazine @EC)
administration
A group of 16 animals were selected of which, half of them were infected with B.
malayi and the other served as control. Four animals each from the control and infected
group were adrmnistered with DEC (6 rng k g body weight orally for 5 days) and grouped as
(I) control (uninfected, Group I), (2) control-DEC treated (Group 2), (3) infected (B.malayi
infected, Group 3) and (4) infected-DEC treated (Group 4). The animals were necropsied
and the ~nfluence of the drug, DEC on the antioxidant enzymes, antioxidants and LPO in
various organs were studied.
4.1.23.1 Superoxide dismutase and catalase
The activity of SOD and catalase were significantly low @<0.05) in all the organs of
B.malayi infected animals (Group 3) as compared with the control (Group 1). Whereas, the
~nfected animals treated with DEC (Group 4) showed a significant w0.05) increase in the
activit~es of these enzymes, except in testes (Figs. 33-36). However, in the lungs, DEC
treatment reduced the increased level of these enzymes. The DEC treahnent did not cause
any s~gniticant changes in the activities of these enzymes in the organs of conbuls (Group 2).
4.1.23.2 Glutathione related enzymes
GST and GPx activities in liver, brain, testes, and heart of B.mnlayi infected animals
(Group 3) wac lesser than that of the control animals (Group 1). And these enzyme levels
i n c d sigruficantly @<0.05) in all the organs of the infected animals treated with DEC
(Group 4), except testes (Fig 37-40). In lungs, the activity of GST rimmed unaffected in
these two groups of animals, but GPx activity was low in infected animals and high in DEC
treated u&ctcd animals (Group 4). These enzymes were unaffected by DEC treatment in
control animals (Group 2).
Fig.33:Effect of 6.malayi on SOD in Liver & Testis after DEC treatment
9 8 7 - 6 , g 5
S 4 s 2 1 0
Testis
Organ
1 Control Control+D Infected Infected+[) 1
Fig.34: Effect of B.malayi on SOD in Brain,Heart & Lungs after DEC treatment
Lungs
Organ
Control C o m l + D Infected Infected+[)
Fig.35: Effect of B.malayi on CAT in Liver & Testis after DEC treatment
T&ie
Organ
I Control Control+D Infected lnfected+D I
Fig.36: Effect of B.malayi on CAT in Brain,Heart & Lungs after DEC treatment
Organ
( Control Control+ D Infected Infected+ D 1
Fig.37: Effect of B.malayi on GST in Liver & Testis after DEC treatment
Liver Testis
Organ
I Control = ControlcD infected f--/ Infected+ D /
Fig.38: Effect of B.malayi on GST in Brain,Heart & Lungs after DEC treatment
1.4
g 1.2
I 1
5 0.8 K
0.6
0.4
0.2
0 Lungs
Organ
Fig.39: Effect of B.malayi on GPx in Liver & Testis after DEC treatment
m 20 0) - 15 9 g 10
il! 5
0 Liver Testis
Organ
Control Control+D Infected Infected+[) 1
Fig.40: Effect of B.malayi on GPx in Brain,Heart & Lungs after DEC treatment
". Lungs
Organ
Control C o m l + D Infected Infected+[)
The GR and G6PDH activities in liver, brain, heart and lungs of B.mlayi infected
animals (Group 3), which were decreased, were restored to nonnal level (significant at
pc0.05) after DEC treatment (&up 4) (Figs. 41-44). However, in testes, despite an increase
in activity, these enzyme levels did not reach the levels found in the testes of the control
animals. In control (Group 2), the administration of DEC, did not bring about any sh~ft in the
activity of these enzymes.
4.1.233 Antioxidants and lipid peroxidation
The decteased levels of GSH, total thiol and ascorbic acid in liver, brain, heart and
lungs of the infected animals (Group 3) were restored to normal levels (significant at F0.05)
after DEC trtatment (Group 4) (Figs. 45-50), however, in lungs, GSH levels did not show
any significant difference. In testes, although there was significant @<0.05) increase in the
antioxidant level on DEC treahnenf it did not match the levels found in the testes of the
control.
The incnased LPO levels in liver, brain and heart of the infected animals (Group 3)
declined significantly QK0.05) aRer DEC treatment (Group 4) (Figs. 518~52). In testes,
though LPO declined after DEC treatment the levels were higher than that of the control
(Group I). In lungs, the LPO, which was decreased in infected animals showed a sipficant
w0.05) increase aRer DEC treatment. The levels of antioxidants and LPO in the organs of
control animals were found to be d e c t e d by DEC treatment (Group 2).
Fig.41: Effect of B.malayi on GR in Liver & Testis after DEC treatment
30
25
8 a 20 - 2 15 C
I 10 I
5
0 Tor*
Organ
Control Control+D Infected Infected+D
Fig.42: Effect of B.rnalayi on GR in Brain,Heart & Lungs after DEC treatment
35
3 30
2 25
9 20 C I 15
f 10
5
0 Lungs
Organ
C o n t r o l Control+D lntected Infected+D
Fig.43: Effect of B-rnalayi on GGPDH in Liver & Testis after DEC treatment
Teatis
Organ
Control Control + D Infected I niected + D
Fig.44: Effect of B-malayi on GGPDH in Brain.Heart & Lungs after DEC treatment
16
UJ 14 5 12
3 10
5 6
2 4
2 0
Lungs
Organ
Control Control+D Infected Infected+ D
Fig.45: Effect of B.maiayi on GSH in Liver & Testis after DEC treatment
Organ
[ Control ~ontrol+ D Infected Infected + D I
Fig.46: Effect of B.malayi on GSH in brain,Heart & Lungs after DEC treatment
35
8 30
25
P 20 3 15
Z 10
5
0 Lungs
Organ
I Control Control+D Infected Infected + D I
Fig.47: Effect of B.malayi on SH in Liver & Testis after DEC treatment
Organ
I Control Control+D Infected Infected+ D I
fig.48: Effect of B.malayi on SH in Brain,Heart & Lungs after DEC treatment
18
16
$ 14
3 12 - g 10
C 8 a 6
8 4
2 0
Hurt Lungs
Organ
Control Control+D Infected Infected + D
Fig.49: Effect of B.malayi on Vit-C in Liver & Testis after DEC treatment
Liver Testis
Organ
Control Control+D Infected Infected + D
Fig.50: Effect of B.malayi on Vit-C in Brain,Heart & Lungs after DEC treatment
25
3 20
15 5 z" lo
5
0 Lungs
Organ
Control Control+D Infected Infected+ D
Fig.51: Effect of B.malayi on LPO in Liver & Testis after DEC treatment
Organ
I Control Control+D Infected Infected+ D 1
Fig.52: Effect of B.malayi on LPO in Brain,Heart & Lungs after DEC treament
12
g 10 3 - m 8 > 5 2
2
0 Lungs
Organ
4.1.3 DISCUSSION
4.1 3.1.1 Toxic oxygen radicals
The last few decades have seen great advances in understanding the parasite induced
host physiology in t m of host defense mechanism especially the role of oxidants (free
radicals) and antioxidant enzymes (Callahan et al., 1988). A free radical is any species
capable of independent existence that contains one or more unpaired electrons. They are
highly reactive and unstable and can injure some biological targets such as proteins, lipids,
carbohydrates and key molecules in membrane and nucleic acids. Moreover, free radicals
initiate autocatalytic reaction whereby, molecules with which they react are themselves
converted into free radical and thus propagate the chain of damage (Halliwell and
Guneridge, 1989).
The free radicals may be initiated within cells during the normal cellular metabolic
processes (Klebanoff, 1974; 1980), by ionizing radiation and certain anti-parasitic drugs
(Docampo and Moreno, 1984) and the host response is also a potent source of oxidants.
Molecular oxygen is a buadical and a relatively unreactive compound. It undergoes a four-
electron reduction to form water. However, it can be metabolized inviw to form highly
reactive derivative oxidants. A series of sequential one-electron transfer yields three reactive
intermediates superoxide anions, hydrogen peroxide and hydroxyl mhcal. When two free
radicals meet, they can join their unpaired electrons to form a covalent bond. When radicals
react with non-radicals. new radicals are generated invivo, which is likely to set off free
radical chain reaction (Halliwell et al., 1995; Thomas, 1995) (Fig. 53). The best studied
biologically relevant fra radical chain reaction is lipid peroxidation.
4.13.1.2 Lipid peroddation
Lipid peroxidation is an important consequence of oxidative cellular damage
(Plea and Witschi, 1976) involved in a variety of diseases and stress (Slater, 1984;
Halliwcll and Guttnidge, 1989). LPO involves the direct reaction of oxygen and p l y
unsaturated fatty acid (PUFA) to form fm radicals and semistable -ides (Tappel,
FIG. 53 FORMATION AND DETOXIFICATION OF REACTIVE
OXYGEN SPECIES IN BIOLOGICAL SYSTEMS. ( Clark et al., 1986)
,Fez' GSH GSSG
0; L-- .v,,%!Gq - h-+* t
616 ' q b i 2
- F e 3 ' i hot4
CELL TOXICITY
O? ' - Superoxide GSH-PX - glutathione peroxidase
SOD - Superoxide dismutase GR - Qlutahone reductase
GSH - reduced glutathione .OH - hydroxyl radical
GSSG - oidised glutathtone S - secondary radical
1973). The two major systems of LPO are non-enzymatic ascorbate induced system
(Ottolenghi, 1959) and enzymatic NADPH induced system (Hochstein and Enister, 1963)
mediated by NADPH cytochrome-c-reductase. Substances such as ascorbate and ferrous
ions, which induce ~ e " to ~ e " , and peroxides enhance LPO (Halliwell and Guaeridge,
1989). LPO decreases membrane fluidity, increases leakiness of the membrane (Jacob
and Lux, 1968) to substances that are normally impermeable, inactivates membrane
bound enzymes (Kesner et a/., 1979) along with the fatty acid and antioxidant depletion.
Damage to the membrane may be subtle and involve only small changes in the
composition of fatty acids, yet often sufficient to greatly increase the susceptibility of the
membrane to oxidative damage (Halliwell and Guneridge, 1985).
Oxygen radicals are generated inside leucocytes enabling them to kill phagocytosed
microorganisms (Babior el al., 1973). Unfottunately for the host, these leucocytes can also
secrete superoxide radicals along with other mediators from their outer membrane into the
surroundings (Nathan and Root, 1977). This indiscriminate and self-inflicting process
contributes to the tissue damage or inflammation (Fig. 54) (Fantone and Ward, 1982; Weiss
and LoBuglio, 1982; Fteeman and Crapo, 1982) and has the capacity to cause tissue damage
in parasite induced disease. Johnson and Ward (1982) reponed that these events occur much
more vigorously when certain receptors on the surface of leucocytes, such as those
responsible to antigen-antibody complexes (Fc receptors) and to complement components
are activated. Thus endothelial damage in h e rat, ~nitiated by immune complexes or C5
activation, can be prevented by depleting animals of neutrophils (Till er al., 1982), infusing
SOD or catalase (Johnson and Ward, 1981; McCormick er a!.. 1981). radical scavengers
(Ward er al., 1983% Fligiel era/.. 1984; Fox 1984) and iron chelators (Ward ef al., 1983b).
Baba ef al. (1989) reported that, in M. nalalensis infected with D. vileae, there was
increase in LPO in the organs such as liver and spleen and decrease in lungs. Red blood cells
infected with P. bwghei have been observed to show five-fold increase in LPO than the
normal (Etkin and Eaton, 1975). Mahdi et a/. (1992) reported increased levels of lipid
W x i d ~ ~ in brain of M. natalemis infected with P. berghei. Elevated levels of LPO
products during malarial infection had been reported (DesCamps er a1.,,1987; Nalr ef al., 3
FIG. 54 THE SEQUENCE OF EVENTS IN FREE RADICAL MEDIATED
LIPID PEROXIDATION (FANTONE AND WARD, 1982)
R'+ PUFA ------+ PIJFA'. R initialion phase
PROTEIN (- puFi.oi -> propagation phase
l'l'i A scavenger' W F A . O H . H,O h e n c conjugates
malonyld~aldchydr crhanc penlanc
Free & ~ Q S ~ndude reduced glutahon+ other hols (for m p l e , protan hols).
vitunin $ d ud flat em^ Scavepyg auyim include supmade h t a s e ,
- d B k u a t ~ a v =
1981), and their increase in human patients with P.falciparum infection are associated with
complications and death. P. vinckei infected RBCs have also been reported to generate
significant levels of malondialdehyde (Clark et al., 1984a, b; Stocker et al., 1985) and it has
been suggested that LPO can be set into motion whenever conditions of increased oxidative
stress or decreased antioxidant defences occur in the cell.
Production of reactive oxygen species in mice infected with P. berghei and P. yeolii
had been reported (Li and Li, 1987; Dockrell and Playfair, 1984). It has been suggested that
HI@ and the reactive oxygen radicals from the respiratory burst have the potential to initiate
LPO, resulting in the formation of toxic aldehydes. Clark ei al. (1987) reported that products
of LPO are toxic and inhibitory against malarial parasite P. falcipam. The products of
macrophage secretions such as oxygenderived free radicals and tumour necrosis factor are
shown to kill the human malarial parasite P. falciparum invitro (Worncraft er al., 1984) and
also initial stages of P. berghei in rodents (Wozencrafl et al., 1985). Hydrogen peroxide
injected intravenously caused a drop in parasitaemia in mice with P. yeolii or P. chaubadi
and can kill P. yeolii and P, berghei maintained invifro (Dockrell and Playfair, 1983). Gharib
et al. (1999) reported a two fold increase in LPO and decreased antioxidants at the site of
pulomatous inflammation in liver infected with Schistosoma mansoni.
ln the present study, it has been found that following infection of B.malayi, LPO
was sigruficantly high in liver, testes, brain and heart of M.natalemis, whereas in lungs it
was low. A negative correlation was observed between the LPO and activity of SOD1
catalase in liver, testes, brain and heart (Figs.55-58, 60, 62-64) and the correlation was
significant 0 . 0 5 ) in liver (SOD I= -0.96). testes (SOD r= -0.97), Brain (catalase -0.89)
and heart (catalase r--0.92) only. The LPO levels, on the contmy, had a positive correlation
with catalase in testes and with SOD in lungs, however it was significant in the lungs (SOD
r= -0.88, p4.05) only (Figs. 59 & 61). This increase in LPO is due to the decrease in the
antioxidant caqmcs and GSH production in these organs. But in lungs LPO operates below
the normal level, although superoxide anions may be produced at a greater rate than in
control, it however, appears to be removed by elevated SOD and catalase (sec. 4.1.2.1.1
Br4.1.2.1.2).
Fig.55:Relationship between change in SOD and LPO levels in liver
SOD
Observed values + Expected values
Fig.56:Relationship between change in SOD and LPO levels in testes
5, I 1
, j ! i r = 0 0 7 p < O m 1 0 y s - 5 4-6 7-r
3 '5
20
25
JO 1 4 5 0 0 5 1 1 5 2 2 s 3
SOD
- Observed values + Expcled values
Fig.57:Relationship between change In SOD and LPO levels in brain
- I
0. - -
r - 4 8 1 p > o m
2.
-2 B-
I 2 s 4 5 e SOD
Fig.58:Relationship between change in SOD and LPO levels in heart
1
0
i l l - -5 1 a -7
-6 -1 0 5 0 0.5 1 1 . 5 2 2.5 3 3 5 4
SOD
( 0 ObSe~0-d values - Expected values I
Fig.59:Relationship between change in SOD and LPO levels in lungs
SOD
= O b w e d values - Expected values
Fig.6O:Relationship between change in catalase and LPO levels in liver
Q I , y--
1 0 1 2 3 4 5 6 7
catalase
/ O b s e ~ e d values + Expected values I
Fig.61 :Relationship between change in catalase and LPO levels in testes
30 I5 10 5 0 5 0
catalase
r Observed values '-- Expected values
Fig.62:Relationship between change in catalase and LPO levels in brain
I I 0 b . 4 V~IU.. -+ ~xpocted values
- -
8 !5
2 5
r = 0 8 0 p<005
y = O 32.032'~
_ - 1
2 1 0 1 2 3 4 5 6 7
catalase
Fig.63:Relationship between change in catalase and LPO levels in heart
1 - 0- ' 1
r=-0 92 pcO05 y = 1 3 7 0 93.x
5 - 6 - 7 - B i
1 0 1 2 3 4 5 6 7
catalase
Observed values - Expected values
Fig.64:Relationship between change in catalase and LPO levels in lungs
,z 1:1* 1 1 0 5 -
0 43 -6 4 -2 0 2 4 6
catalase
I Observed vdues + Expected values I
The LPO altered during infection of B.malayi was brought back to normal levels after DEC
treatment and this has happened due to the restoration of the activity of antioxidant enzymes
and GSH to normal levels as observed in a further experiment. In testes, the LPO was
remarkably high compared to controls and even after DEC treatment, did not reach normal
level. This may be due to the irreversible tissue damage as evidenced in histopathological
studies (sec. 4.4.2.2).
4.1.3.2 Superoxide dismutase and catalase
The cells are rich in the antioxidants such as GSH and an antioxidant enzyme like
catalase, SOD. GR and GPx and contains a proteolytic system that can hydrolyze oxidatively
modified protein (Halliwell and Gutteridge, 1989). Disrnutation of superoxide anion is
catalysed by SOD, which yields Hz@, and it is W e r decomposed by the enzyme catalase
and glutahone peroxidase. This reaction plays a major role in protecting the cell membrane
from H 2 q (Jacob er a/., 1%5; Panicker and Zyer, 1969), but a decrease in the activities of
SOD, catalase. GPx, GR and GSH level can exacerbate LPO.
Studies conducted on D.viteae infected animals (Baba et a/. , 1989), indicated that the
act~vity of SOD decreased in liver and spleen, but increased in lungs. Red blood cells
parasitized by P.krghei (Fairfield et a/.. 1983; 1986) and P. vinckei (Stocker et al., 1985)
contained less SOD and catalase than the RBC's of uninfected. Altered levels of SOD have
also been nponed in patients with P. falcipanun mfection and in mice with P. berghei and
P. vinckei infection (Areekul and Boonme, 1985, 1987; Suthipak el nl., 1982; Stocker et al.,
1985; Fairfield el a/., 1988). Areckul and B o o m (1987) had shown that higher
parasitaemia of RBC's, due to Pjalcipnun is associated with higher SOD activity
compared to the uninfected ones. Fairfield et a/ . (1983) reported that P. berghei devoid of
endogenous SOD adopts e y h q t e SOD for protection against the deleterious effect of
superoxide radicals and consequently reduces the host SOD level. SOD and catalase were
decnesed significantly in P, v i m malaria in patients (Mathews and Selvam, 1991). SOD
showed restoration of enzyme activity while. catalase activity was increased significantly
after primaquine trratmcnt.
Catalase had been reported to be responsible for the detoxification of Hz@ (Brenner
and Alison, 1953; ~icholis, 1965). Small quantity of erythrocyte catalase was observed to
exist in inactive form viz., catalase complex I1 (Leibowitz and Cohen, 1968). The conversion
of complex I1 to active form is dependent on the reducing agent NADPH (Eaton el al.,
1972). Under conditions of increased oxidative stress, it is likely that more NADP rather
than NADPH is formed, which is required for the activation of catalase. The decreased
NADPH and G6PDH leads to reduced catalase activity (Eaton er al., 1972). The reactive
oxygen radicals can also inhibit the activity of catalase and GPx as had been observed by
Hodgson and Fridovich (1975) and Searle and Wilson (1980).
In M nalulensis infected with D. viteae, decreased activities of catalase and
xanthine oxidase and increased LPO were observed in liver and spleen (Batra el al.. 1989).
And on the other hand, in lungs the elevated activity of xanthine oxidase and catalase
prevented the LPO. Catalase activity was also found to be low in P, vivm (Mathews and
Selvam, 1991) and P. ful~ipanun (Areekul and Boonme, 1987) infected human erythrocytes,
and in mrce erythrocytes infected with P. berghei (Nair e! al., 1984) and P. Vinckei mckard-
Maureau el al.. 1975).
In the pment study, in the liver, brain, testes and heart of the infected animals, the
actlvrty of catalase increased initially and declined later. However, in lungs, its activity
rncreased wrth the p r o p i o n of the parasite development. Fielden et al. (1974) and Bray
(1974) observed that SOD activity never decreased in the presence of catalase, which
suggested that the reaction product H 2 q inactivated SOD (Yamakura and Suzuki, 1986).
Kono and Fridovich (1982) demonsmted that catalase was inhibited by superoxide anion
and concluded that SOD and catalse are mutually acting protective set of enzymes. In the
present study, a positive correlation was obsewed between the activity of SOD and catalase
In liver, brain. heat and lungs (Figs. 65,67-69), and the relationship was significant w0.05)
In brain (14.94) and heart ( ~ 0 . 9 7 ) only. Compared to this, in the testes, a negative
cornlation was observed W e e n the activity of SOD and catalase, and it was not
significant. The deed SOD and catalase activity in the organs of B. malayi infected
animals may be due to the inhibition by reactive oxygen radicals released and non- &
availability of NADPH. This is & m ~ e d between 60 and 120 days post rnfection, which is
Fig.65:Relationship between change in SOD and catalase levels in liver
I Observed values -+- Expected values I
a-
Fig.66:Relationship between change in SOD and catalase levels in testes
7. 6-
3
r = 047p>OOS
10-
154 I ,
1 4 5 0 0 5 1 1 5 2 2 5 3
SOD
m
r e 0 27 D>O M y-54-67.X
I I Ohewod values --c Expbcted values
9 1: +-
+
I
1, 1 0 1 2 3 4 5 e
SOD
Fig.67:Relationship between change in SOD and catalase levels in brain
1-091 p < o m
y c 147+1 U.1
I
0 1 2 S 4 5 6
SOD
Fig.68:Relationship between change in SOD and catalase levels in heart
6 - r=OS? p<005
5 - y = l 08+1 86.x
- m 3- -
1 - 0 5 0 0 5 1 1 5 2 2 5 3 3 5 4
SOD
1 - O b S e ~ e d values -+- Expected values /
Observed values -+- Expected values --
Fig.69:Relationship between change In SOD and catalase levels In lungs
6
2
3 0 - f 2
1
I r - 0 71 p=-0 05
y = 3 15+044.x
m I
-4 -8 25 :! 20 I) 15 10
1
SOD
the period by which, the parasite attains full development and gets lodged in the organs. In
lungs, conhary to other'organs, the levels of SOD and catalase showed increasing trend
throughout the study period indicating that more amount of NADPH is formed by the
restoration of G6PDH (sa. 4.1.2.1.6), even though the parasite burden is higher than that
observed in other organs.
The activity of SOD and catalaw which were altered in all the organs of B.ma1ayi
infected animals were restored to normal levels after DEC treatment. And this may be
associated with concornittarit generation of NADPH by the increase in G6PDH activity after
DEC treatment as observed (sec. 4.1.2.3.2).
4.133 Glutathione and glutathione related enzymes
GSH plays an important role in protection of cells against oxidants in most
organisms (Bryant and Behm, 1989). Depletion of GSH leads to lethal conditions and
accumulation of o x i d i i glutathione (GSSG), which is also harmful to cells, because it
forms mixed disulphides with proteins and other molecules containing thiol groups. The
concentration of GSH in tissues is regulated by its generating enzyme GR from oxidised
glutathione. The utilising enzymes GPx and GST convert the reduced state (GSH) to
oxidized form (GSSG) during the detoxification of the radicals. The NADPH r e q d for
GR activity is regenerated by the enzyme G6PDH (Halliwell and Gutteridge, 1990).
Jakoby et al. (1976) reported that GSH is capable of protecting cells from oxidant
s tms owing to its antioxidant and nucleophilic charactenstics. It helps in maintaining the
integrity of nd cells (Fegler. 1952) particularly when exposed to oxidant stress due to drug
or malarial infection (Allen and Jandl, ]%I) by protecting hemoglobin and other thiol
containing proteins from denaturation.
GPx plays an important role in the detoxificat~on of Hz&, organic hydroperoxides or
lipid peroxides (Cohen and Hochstein, 1963: Chnstopherson, 1%8; 1969; Flohe, 1982).
GSH and NADPH are required in awuate amount for GPx activity @*their depletion.
results in its reduced activity (Condell and Tappell, 1983). Also that GPx is inactivated under
severe oxidative sees^. .
In P. knowlesi infected monkey, the activity of GSH had been reported to be
decreased (Fulton and Grant, 1956; Fletcher and Maepith. 1970). Bhattacharya and
Swarupmithra (1987) reported a decline in the etythrocyte GSH In P.vivlu infected patients.
P. knowlesi infection resulted in elevation of hepatic stress in monkeys, besides affecting the
antioxidant enzymes of the host. Gpx activity was found to be low in P. vinckei infected
mice erythrocytes by Stocker et al. (1985) and in P. viva by Mathews and Selvam (1993)
and Sarin (1 993). In studies with P. falcipanrm and P. viva infected RBC's GR activity was
also found to be reduced (Stocker et al., 1985; Kamchogwongpaisan el al., 1989). Litlov el
al. (1981) reported that in the absence of GSH, loss of GR activity is seen. GST is one of the
GSH utilizing enzymes and reduction in GSH leads to the decreased activity of GST
(Srivastava el al., 1995).
Picard- Maureau er al. (1975) had observed decrease in the activity of G6PDH of
P.vinckei parasitized mice erythrocytes and a similar trend was reported in P. bergei infected
mice (Nair et 01.. 1984; Grindberg and Soprunov, 1983) and in the RBC's of patients
afflicted w~th malaria (Mathews er al., 1991).
Srivastava er al. (1991; 1992) and Clark et al. (1986; 1989) reported an increased
oxidative damage during malaria infection and attributed this to decreased GST level.
Srivastava er al. (1995) had shown that P berghei infection in M.naraletzsis reduced the
activity of GST in the hepatic mitochondria and microsome and the GST level was brought
back to normal aAer chloroquine treatment. Mathews and Selvam (1993) reported decreased
GST activity in P. vivor infected human erythrocytes.
In the present study, GSH level decreased in liver, brain, testes and heart of the
B,mafayi infected animals, but in lungs it showed an increasing trend. A negative correlation
was observed between the activity of GSH and LPO in all the organs (Figs. 70-74) and it was
significant w 0 . 0 5 ) in liver (I=-0.88), testes (I=-0.95) and heari (r--0.88) only. Similarly a
positive cornlation was observed between the activity of GST and G S I ~ ip all the organs
(Figs. 75-79) and it was significant wO.05) in liver ( ~ 0 . 9 3 , testes (I= 0.94) and brain
( ~ 0 . 9 8 ) only. The activity of GRI G6PDH had a positive correlation with GSH activity in all
the organs (Figs. 80-89) and it was significant (~4.05) in testes ( ~ 0 . 9 6 , 0.93), brain
( ~ 0 . 9 8 , 0.98) and heart ( ~ 0 . 9 8 , 0.88) only. There was positive and significant ( ~ ~ 0 . 0 5 )
correlation between the activity of GPx and GSH in all the organs (Figs. 90-94), such as liver
(r-0.9), testes (r=0.98), brain (r= 0.97), heart ( ~ 0 . 9 4 ) and lungs (F0.9). In the present study,
the level of NADPH regenerating enzyme, G6PDH was found to be low and this condition
might have resulted in the non-availability of NADPH. The decrease in GR (GSH generating
enzyme) activity is due to the non-availability of the reducing equivalent NADPH required
for the conversion of oxidized glutathione to reduced glutathione which is the substrate for
this enzyme (Halliwell and Gutteridge, 1990). The decrease in GPx and GST (GSH utilising
enzymes) is related to the reduction of GSH levels. Thus the decrease in the GSH generating
and utilising enzymes reduced the GSH level in the B.malayi infected animals. However, in
lungs the availability of G6PDH in plenty had increased the GSH level.
Following DEC administration the GSH level in all the organs of the B.malayi
infected an~mals significantly ~ncreased and thls is due to the restoration of the activity of the
enzymes G6PDH. G R GST and GPx as stated above.
4.1 3.4 Total thiols
Loss of membrane suphydryl groups renders the membrane increasingly susceptible
to LPO with increase in membrane permeability, leading to colloid osmotic hemolysis and
reticuloendothelial entrapment (Jacob and Jandl, 1962). The depletion of membrane
sulphydryl attached to fatty ac~d double bonds renders them increasingly vulnerable to
pemxidative cleavage, which in turn leads to membrane dissolution (Jacob and Lw, 1968).
F1g.70:Relationship between change in GSH and LPO levels in liver
- ,=pen p < o w
y = l 17 1 07% :I-. 8
10
12 0 2 4 6 8 10
GSH
[ r Observed values + Expected Values 1
Fig.71 :Relationship between change in GSH and LPO levels in testes
30 0 2 4 6 8 7 0
GSH
I - Observed values -- Expected values /
Fig.72:Relationship between change in GSH and LPO levels in brain
5 - 0. - I 5 .
,1082 p>om I 10- y r 2 7 0 3 38'r
I 15. g 20- 25.
30.
35.
I 40.
4 b t 4 2 1 6 (I 10 l ? 14
GSH
Fig.73:Relationship between change in GSH and LPO levels in heart
.. I -7
1 0 1 2 3 4 5
GSH
I Observed values + Expected values 1
Fig.74:Relationship between change in GSH and LPO levels in lungs
GSH
I Observed valuer + Expected values I
Fig.75:Relationship between change in GST and GSH levels in liver
12, - y = 089+802-x
I 0 U) Q 4
2
0 - + - - -
202 0 0 2 0 4 0 6 0 8 1 1 2 I d
GST
I Observed values + Expected values :
Fig.76:Relationship between change in GST and GSH levels in testes
r=004 ~ ~ 0 0 5
y = - 0 6 4 4 7 1-x x- , -- 2 0 0 2 0 4 0 6 0 8 1 1 2 1 4
GST
I Observed values + Expected values
Fig.77:Relationship between change in GST and GSH levels in brain
14
12 r-096, p < O M
10
04 - . I 0 0 1 0 2 0 3 0 4 0 1 0 6 0 7 0 8 0 8
GST
Fig.78:Relationship between change in GST and GSH levels in heart
-2 4 005 0 1 0 15 0.2 0 25 0 3 C
GST
[ Observed values - Expected values /
Fig.79:Relationship between change in GST and GSH levels in lungs
2 5 I -
0 4 -0 05 0 0.05 0 1 0
GST
I 9 Observed values - Expected values 1
Fig.80:Relationship between change In GR and GSH levels in liver
$
0 4
2
0 .. 2
2 0 2 4 6 8 10 12 14 16 18
G R
I Observed values --t Expected values I
Fig.81 :Relationship between change in GR and GSH levels in testes
I Observed values + Expected values
I Oboowad values + Expected values
Fig.82:Relationship between change in GR and GSH levels in brain
10-
14.
12-
10'
5 0.
a 6 .
4 -
2-
0,
r=O98. pcO M y= 1 2 + 1 Max
2 0 2 4 0 E 10 12 1.4
G R
Fig.83:Relationship between change in GR and GSH levels in heart
- Observed values + Expected values
Fig.84:Relationship between change in GR and GSH levels in lungs
- Obsefved values + Expected values
Fig.85:Relationship between change in G6PDH and GSH levels in liver
I r Observed values + Expected values (
Fig.86:Relationship between change in GGPDH and GSH levels in testes
( I Observed values -+- Expected values I
Fig.87:Relationship between change in G6PDH and GSH levels in brain
Fig.88:Relationship between change in '
G6PDH and GSH levels in heart
I - Obsewed vdues + Expected values /
Fig.89:Relationship between change in GGPDH and GSH levels in lungs
I = Observed values + Expected values /
Fig.9O:Relationship between change in GPx and GSH levels in liver
GPx
I Observed values + Expected values
Fig.91 :Relationship between change in GPx and GSH levels in testes
I m Observed values - Expected values
Fig.92.Relat1onship between change In GPx and GSH levels In bra~n
V) (3 :; 6
r - 0 0 7 p < O M
"-2 CU* I 81.r -. 1 0 1 2 3 4 5 6 7
G Px
Fig.93:Relationship between change In GPx and GSH levels in heart
61 1
I Observed values + Expected values I
5-
4-
1 3- (I) 0 2-
1 - 0-
1 -
Fig.94:Relationship between change in GPx and GSH levels in lungs
y=-0 41 +O 79.x
0 , --A
-0 5 0 0 5 1 1 5 2 2 5 3
GPx
1 2 3 4 5 6
GPx
I O b s e ~ e d values - Expected values I
A reduction in the total thiol contents was observed in the present study in different
organs of B.malayi infected animals. A significant w . 0 5 ) positive comlation was
observed between thiol status and GSH levels in all the organs (Figs. 95-99) such as liver
( d . 9 6 ) , testes ( ~ 0 . 9 ) . brain ( ~ 0 . 9 8 ) . heart (r=0.96) and lungs (I4.89). (Kosower et al.
(1982) had shown a direct link between the thiol status of the membrane and cellular GSH.
This shows that the function of GSH is to serve as a reducer of membrane protein
disulphides and to avert membrane thiol oxidation. The decreased GSH concentration
observed in the present study had contributed to decreased thiol status, while in lungs the
availability of GSH had increased the thiol level. The decreased total thiol levels in the
B.malayi infected animals were restored to the normal levels after DEC therapy, which is
due to the restoration ofGSH levels (sec. 4.1.2.3.2).
4.13.5 Ascorbic acid
The antioxidant property of ascorbic acid is often associated with its ability to
regenerate vitamin E h m vitamin E radical (Niki eta/., 1984). Nishlkimi (1975) had shown
that ascorbate reacts with superoxide and converts it to Hz@. Ascorbic acid levels in plasma
were found to be decreased due to P.vivar infection (Irwin and Hutchins, 1976; Frei er a/.,
1988). Tappel (1969) and Grimble and Hughes (1967) reported that GSH is required for the
conversion of dehydroascorbate to ascorbate. In the present study ascorbic acid levels were
found to be low in liver, brain, testes and heart except for lungs of the B.malayi infected
animals, which is due to the alteration in GSH level during infection. The ascorbic acid level
and GSH had positive comlation in all the organs (Figs. 100-104) and it was significant
w0.05) in liver (r=0.99), testes ( ~ 0 . 8 9 ) ~ brain (r= 0.88) and heart (14.97) only. And the
restoration of the ascorbic acid levels observed in the infected animals after DEC treatment
is due to the restored GSH levels (sec. 4.1.2.3.2).
Fig.95: Relationship between change in total thiol and GSH levels in liver
r = O O B p < O M
Y = 1 45+2 1 4 - r
0 4
0 - <kc Total th~o l
1 - Observed values --c Expecled values )
Fig.96:Relationship between change in total thiol and GSH levels in testes
l o '
8- r = O Q p < O M
y - 0 12+2 G5-r 6 -
8 WJ 4 - 0
2 - OQ
I
2i 0.5 1 1 5 2 2 5 3 3 5 4 4 5 5
Total th~ol
I Observed values -+- Expected values
Fig.97:Relationship between change in total thiol and GSH levels in brain
3 4 ,
l a - 7 - 0 9 6 p-005
$0- "- a 1 8 - 3 I6.r
0 - I UY 0 - * 4.
2 - 01
"g o i i 1 s 2 2 5 3 3 5 4 4 5
Total thlol
Fig.98:Relationship between change in total thiol and GSH levels in heart
1 4 -2 1 0 1 2 3 4 5
Total thiol
I - Observed values + Expected values I
Fig.99:Relationship between change in total thiol and GSH levels in lungs
2 5 -
2- r=oes PC005
y - 0 3 5 + 0 3 7 ' x
I 15'
% I -
0 5 -
1 2 3 4 5
Total thiol
- Observed values - Expected values
Fig. 100:Relationship between change in ascorbic acid and GSH levels in liver
12
y=035+1 50.r
6Y 0 4
2
0
2 2 1 0 1 2 3 4 5 6 '
A s c o r b l c a c l d
1 9 Observed values -+- Expected values I
Fig.101 :Relationship between change In ascorbic acid and GSH levels in testes
10 - I 8- ,=Om pcODJ
y = 2 11+0m.x 6-
I 6Y 4 - 0
I ,
0 2 4 6 6 10 12 ' 4
Ascorb~c ac~d
/ - Observed values -+- Expected values (
Fig.lO2:Relationship between change In ascorbic acid and GSH levels in brain
Ascorbic a c l d
1 - Ota.wed values - Expected ~~~~~~~1
Fig. 103:Relationship between change in ascorbic acid and GSH levels in heart
Ascorbic acid
9 Observed values +- Expected values
Fig. 104: Relationship between change in ascorbic acid and GSH levels in lungs
Observed values - Expected values
CHAPTER 4.2
CHANGES IN THE ACTMTY OF MEMBRANE BOUND ENZYMES AND
HAEMATOLOGICAL PARAMETERS IN MASTOMYS NATALENSIS INFECTED
WITH BRUGlA MALAY/
4.2.1 MATERIALS AND METHODS
M. natalensis of the same age group (6-8 weeks) and weight (35-40 grn) were
divided into two groups, one served as the control while the other was infected with B.
rnalayi infective larvae as described earlier. At 0, 30, 60, 90 and 120 days of post-
inoculation, brain homogenate of the animals were prepared as described earlier and used for
the determination of the activity of different enzymes. Blood samples were collected at 0,30,
60, 90 & 120 days post infection and used for haematological and biochemical
investigations.
The erythrocyte membrane was isolated according to the procedure of Dodge et al.
(1%3). Blood plasma was removed and the remaining packed cells were washed three times
with isotonic phosphate buffer, pH 7.4. The washed RBC suspension was haemolysed with
hypotonic buffer (20 milliosmolar, pH 7.2) in the ratio of buffer: cells of 14:l. The ghost
cells were sedimented in a high speed refi-igerated centrifuge at 20,Wg for 40 minutes. The
supernatant was decanted carefully and the ghost button was resuspended by swirling.
Sufficient buffer of the same strength was added to reconstitute the original volume. The
mtio of cells to washing solution was approximately 1:3 by volume. The ghosts were washed
three times subsequent to haemolysis. The supernatant after the last wash was either pale
pink or colourless. The pellet of erythrocyte membrane was suspended in 10 ml of 0.32 M
sucrose solution and hornogenised. Aliquots of this homogenate were used for enzyme
assays and estimation of protein.
4.2.1.1 N a y ATPase
N a y ATPase activity was determined as per the method of Bonting (1970).
Reagents
1. Tris-HCI buffer : 184 mM, pH 7.5
2. Magnesium sulphate : SO mM
3. Potassium chloride : SO mM
4. Sodium chloride :600mM
5. EDTA : I m M
6. ATP : 40 rnM
One ml of Tris buffer, 0.2 ml each of all the other reagents listed above were mixed
together such that the final volume of 2.0 ml contained 92 mM Tris buffer, 5 mM
Magnesium sulphate, 60 mM sodium chloride, 5 mM potassium chloride, 0.1 mM EDTA
and 4 rnM ATP. After 10 minutes equilibrium at 3PC in an incubator, reaction was started
by the addition of 0.1 ml homogenate. The assay medium was incubated for 15 minutes and
then the reaction was stopped by the addit~on of 1.0 ml of 10% TCA.
The amount of phosphorous liberated by the enzyme from ATP was estimated by the
method of Fiske and Subbarow (1925). And the enzyme activity was expressed as pmoles of
phosphorous liberated/min/mg protein.
4.2.1.2 ca2+ ATPase
The activity of ca2' ATPase was estimated according to the method of Hjerten and
Pan (1983).
&IrgentS I . Tris-HCI buff^ : 125 mM. pH 8.0
2. Calcium chloride : 5OmM
3. ATP : 1OmM
To the reaction mixture containing 0.1 ml each of calcium chloride, ATP and buffer,
0.1 rnl of 1:20 diluted homogenate was added and incubated at 37C for 15 minutes. Then
the reaction was arrested by the addition of 0.5 ml of 10 % TCA. The amount of
phosphorous liberated From the substrate was estimated by the method of Fiske and
Subbarow (1925) and the enzyme activity was expressed as p o l e s of phosphorous
liberatediminlmg protein.
4.2.13 M~'' ATPase
The activity of M$ ATPase the enzyme was estimated by the method of Ohnishi et
al. (1982).
Reagents
1 .Tris-HC1 buffer : 375 mM, pH 7.6
2. Magnesium chloride : 25 mM
3. ATP : IOmM
The assay was initiated by the addition of 0.1 ml of 1:20 diluted homogenate to the
reaction mixture containing 0.1 ml of water and 0.1 rnl of each of the above reagents.
Incubation was carried out at 3 7 C for 15 minutes and then the reaction was terminated by
the addition of 0.5 ml of 10% TCA. The amount of phosphorous liberated from the substlate
was estimated by the method of Fiske and Subbarow (1925) and the enzyme activity was
expressed as p o l e s of phosphorous liberatedimidmg protein.
4.2.1.4 Acetylcholinesterase
Acetylcholinesterase was estimated by the method of Hestrin (1949)
Reagents
1. Acetylcholine bromide : 0.1 M
2. Sodium chloride : 1 M
3. Magnesium chloride : I M
4. Tris-HCI buffer : 0.5 M, pH 7.5
5. EDTA : 0.2 M
6. Cocktail : Mix 13 ml of 1.0 M sodium chloride, 2 rnl of 1 M
magnesium chloride, 10 ml of 0.5 M Tris-HC1 and I0 rnl of 0.2 M EDTA.
7. Hydroxylamine hydrochloride : 2 M
8. Sodium chloride : 3.5 M
Mix reagents 7 and 8 before use in 1: 1 ratio viv (prepare freshly)
9. Hydrochloric acid : 6 N
10. Fenic chloride : 0.37 M in 0.1 M HCI
1 1 . Reaction &urn : Mix 17.5 rnl of cocktail with 2.0 ml of 0.1 M
acetylcholine chloride and 5.5 ml of distilled water. Tlus reaction medium gives the final
concentration of 130 mM sodium chloride, 20 mM magnesium chloride, 50 mM Tris-HCI,
0.2 mM EDTA and 4 mM acetylcholine chloride.
To the reaction mehum of 0.5 ml, 0.1 rnl of tissue homogenate and 0.4 ml of
distilled water were added and the reaction mixture was incubated at 3% for 30 minutes.
Then the reaction was terminated by addmg 2.0 ml of hydroxylamine hydrochloride and
sodium chloride mixture. The contents were mixed by vortex mixer and after one minute, 1
ml of HCI was added and mixed. Then 1 ml of femc chloride was added and absorbance was
read at 540 nm. The activity of the enzyme was expressed as pmoles of acetyl choline
bromide utiliscd/min/mg protein.
4.2.1.5 7-glutamyl transpeptidase
yGTP was assayed according to the method of Orlowski and Meister (1965) and
modifid by Rosaki and Rau (1972).
Reagents
1. L-y-glutamyl pnitmkilide : 30.3 mg dissolved in 10 ml of distilled water.
2. Tris-HCI buffer : 0.1 ml, pH 8.2
3. Glycyl glycine : 13.2 mg dissolved in 10 ml of distilled water.
4. Acetic acid : 10%
5. Standard : 13.8 mg of p-nitroaniline (recrystallised) (ImM) in
100 ml of distilled water.
The tissue homogenate (0.5 ml) was added to the reaction mixture containing 0.5 ml
y-glutamyl pnitroanilide, 2.2 ml of glycyl glycine and 1.0 ml of buffer. AAer incubation for
30 minutes at 3?C, the reaction was terminated by the addition of 1.0 ml of 10% acetic acid.
The amount of pnitroaniline liberated in the supematant was measwed, as the difference In
the optical density at 410 nm between samples, with and without substrate. The substrate
incubated in the absence of enzyme under the same conditions was used as a reference blank.
Optical density of solution of p-nitroaniline in the range 0.005-0.02 p l e s served as
standard cwve for aniving at the amount of product formed. The activity of the enzyme was
expressed as p o l e s of pnitroaniline liberated/midmg protein.
4.2.1.6 Alkaline phosphatase
Alkaline phosphatase was assayed by the method of ffing (1965) using disodium
phenyl phosphate as substrate.
Reagents
1. Carbonate buffer : 0.1 M, pH 10.0
2. Disodium phenyl phosphate : 0.01 M
3. Magnesium chloride :0.1 M
4. Sodium carbonate : 15%
5. Standard : 100 mg of redistilled phenol in 100 ml water.
To 1.5 ml of carbonate buffer, 1.5 ml of disodium phenyl phosphate, 0.1 ml of
magnesium chlorideand 0.1 ml of 1:20 diluted tissue homogenate was added and incubated
for 15 minutes. The reaction was stopped by adding 1.0 ml of 10 % TCA. The contents were
then centrifuged and the residue was discarded. To the supernatant, 1.0 ml of sodium
carbonate solution and 0.5 ml of Folin's reagent was added and the absorbance was read at
640 nm after 10 minutes. The activity was expressed in terms of pmoles of phenol
liberatedimg protein4 5 minutes.
4.2.1.7 Haemoglobin
Haemoglobin was estimated by the cyanmethaemoglobin method of Drabkin and
Austin (1932).
Reagents
1. Fenicyanide-cyanide reagent: This was prepared by dissolving 200 mg potassium
fenicyanide, 50 mg potassium cyanide and 140 mg potassium dihydrogen phosphate in a
litre of water.
2. Cyanmethaemoglobin standard: Purchased from Span Diagnostics Pvt. Ltd., Surat, India.
This was kept in the dark at 4'C. It had an equivalent hemoglobin concentration of 60 mg%.
Twenty p1 of blood was added to 4.0 ml of the fenicyanide-cyanide reagent. This
was allowed to stand for 15 minutes and read against a reagent blank at 540 nm. The
standards were diluted in fenicyanide-cyanide solution to obtain a range of concentrations
and read in the same manner.
Blood hemoglobin values were expressed as &dl.
4.2.1.8 Total RBC counts
Total RBC counts was determined according to the standard procedure (Dacie and
Lewis, 1975).
The method involves an accurate dilution of a measured quantity of blood with a
fluid, which is isotonic with the blood, which will prevent coagulation.
The RBC pipette was filled with blood to the 0.5 mark and the diluent was filled upto
101 mark to make it to 1 :200 dilution. The pipette was shaken for three minutes and the first
two drops were discarded. The cover slip was placed on the haemocytometer counting
chamber and a few drops of diluted blood were filled and left to settle for one minute and
then counted under a light microscope. The total RBC was calculated using the following
formula:
RBC/cu.mm = cells counted x 5 (115 cm2 counted) x 10 (depth) x 200 (dilution factor).
4.2.1.9 Total leucocyte count
Total leucocyte count was estimated by the haemocytometer method (Miale, 1972).
Blood was diluted with a fluid that lyses the non-nucleated erythrocyte precursors. If
the blood smear showed nucleated erythrocytes, the cell count was corrected to the true
leucocyte count according to the following formula:
Corrected count = Observed count x 100
100 + % nucleated erythrocytes
The WBC pipette was filled to the 0.5 mark with blood and diluted to the 11 mark
with I .Ph HCl. This made a 1:20 dilution. The pipette was shaken for three minutes and the
lint two drops were discarded. The haemocytometer chamber was filled with diluted blood
and left to settle for one minute.
Lcucaytes present in the four large c o w squares (1 sq.mm each) were counted and
calculated by tbe following formula:
WBC/cu.mm = Cells counted x IOfdeotb) x 2Mdilution factor)
4(sq.mm. counted)
4.2.1.10 Differential leucocyte count
The leucocyte differential count was detenined by Leishman's staining method
(John, 1972).
Reagents
1. Leishrnan's stain : 0.2 g of Leishman dye was dissolved in 100 ml of acetone free
methanol at 50°C for 15 minutes with occasional s w n g , then cooled and filtered.
2. Buffered water : 0.066 M Sotenson's phosphate buffer was made upto 50 ml, pH 7.0
and mixed with 3.89 ml of KH2P04 (9.1 gm4) and 61.1 ml NazHPO4 (9.5 gm4) to I litre
water
A drop of blood was placed on a clean slide and a spreading slide was placed at
an angle on the slide and moved forward to make a smear of 3-4 cm in length and air-dried.
The slide was covered with stain for 2-3 minutes and the stain was diluted by adding drops
of buffered water and left for 5-7 minutes. The slide was washed with water, dried and
examined under a microscope.
4.2.2 RESULTS
4.2.2.1 Membrane bound enzymes of brain.
The changes in the activity of the membrane bound e v e s of the brain are
presented in the Figs. 105-107. The activ~ty of all the enzymes studied ( N a y ATPase,
M ~ ~ ' ATPase, Ca2' ATPase, yGTP, acetylcholinesterase and alkaline phosphatase),
increased continuously from day 0 to day 120 in the control animals, whereas in the
B.malayi infected ones there was a significant (pC0.05) decrease from 60 days to 120 days.
43.2.2 Membrane bound enzymes of RBC
The activity N~'K' ATPase, Ca2' ATPase and h4g" ATPase in the infected animals
1- slightly from 0 day to 30 days and d e d the~fter as aglost the continous
Fig. 105: Effect of 6.malayi on N~+K+ and ~ d % ~ ~ a s e in brain of M. natalensis 0 10,
0 14-
0 12-
! 01- -
g ooe-
= ow... O M -
0-3 0 30 W 90 120
Days of ~nfect~on
Fig.lO6:Effect of 6.malayi on M & P ~ S ~ & Ach-est in brain of M. natalensis
0 14-
0 12-
01-
ow.
0 M-
0 30 W PO 120
Days of lnfecbon
Fig.107: Effect of B.malayi on GTP and AP in brain of M. natalensis
3 5-
Days of tnfectton
I - GTP.N -C OW-I - AP-N * AP-I ]
increase in the control animals (Figs. I08 & 109). And the enzyme, acetylcholinesterase was
found to decrease s ~ ~ f i c a n t l ~ (p4.05) 6om 0 day to 120 days.
4.2.23 Haematological parameters.
The effect of parasitic development on some haematological parameters was studied
(Figs. 110-1 13). The hemoglobin levels and RBC counts in the infected animals were fokd
to fall within the normal physiological range (Appendix). The total WBC count in the
infected animals was significantly w0.05) lower than the normal values.
A significant decrease w0.05) was observed in the absolute count of lymphocytes
after 80 days post infection, while the eosinophil and neutrophil count increased significantly
(F0.0 I ).
4.23 DISCUSSION
4.23.1 Membrane bound enzymes of brain.
Membrane bound enzymes such as Na'K' ATPase, M ~ ~ * ATPase, ca2* ATPase,
plays an important role in the maintenance of the ionic gradient between the ~nbacellular and
exhacellular compartments of the cell (Trump et al., 1979). Acetylcholinesterase is involved
in the banmission of nerve impulses. N a y ATPase plays an important role in the active
eansport of ~ a ' and K* ions (Mahaboob Basha and Nayeemunnisha, 1993), conduction of
nerve impulses and synaptic function in the brain (McIlwah 1969; Sweadner, 1979). M~~
ATPase and ca2' ATPase have been found to regulate ionic pumps in the CNS (Farber,
1982).
In the present study, the activity of NaX' ATPase, M ~ " ATPase and ca2' ATPase
of the CNS d a e a x d to significant levels. ATPases depend on lipids and thiol groups to
maintain their structure and function (Gamer et a/. , 1983; h h h , 1980; Shalev et al., 1981).
Oxygen fne radical production during B.mahyi infection leads to oxidation of thiol groups
(SCC 4.1.2.1.8). This decrease in Na'K' ATPase may be aseociated to l$t decrease in the thiol
level as stated earlier. Thc reduced activity of N~'K' ATPase leads to decreasg: in sodiwn
9 0
Fig.! 08:Effect of B.malayi on ~ a ' K' and caMATPase in RBC of M.natalensis
0.024 I 0 30 60 90 120
Days of infection
- Na-N - Na-t - Ca-N -- ca-l
Fig.lO9:Effect of B.malayi on M ~ % T P ~ S ~ and Ach est in RBC of M.natalensis
Days of infection
- -Mg-N -+- Mg-l - Ach est-N - Ach est-l I
Fig.110: Effect of 6. malayi on Hb and RBCofM.natalensis
Days of infection
I - H ~ J - N - H W - RBC-N - RBC-I 1
0 7 T I 0 30 60 90 120
Days of infection
Fig.111: Effect of B. malayi on Total WBC of M.natalensis
6000 -,
r- TOW WBC-N - Total WBC-I 1
5000 - Q, 3 4000- - 2 C Q 3000-
g 2000-
- + I, -
ill i
Fig. 1 1 2: Effect of 8. malayi on Lymphocytes of M.natalensis
90 -, 1 801 70 -
0, 3 60- - m > 50-
40-
30-
20 -
10- E
0 0 30 60 90 120
Days of infection
Fig.113: Effect of B. malayi on Neutrophils & Eosinophils in Mastomys
7"
40 - 35-
0, 3 30- - m > 25-
5 15-
10-
5 -
20" 4 B
- - -.
OF 0 30 60 90 120
Days of infection
efflux and thereby alter the membrane permeability, and also likely to affect the transport of
Na' ions as well as nerve impulses and synaptic functions of the brain as reported above.
The decrease in ca2' ATPase and Mg2' ATPase activities is due to peroxidation of the
membrane lipids (sec 4.1.2.2). The lowered activity of Mg2' ATPase and ca2' ATPase
observed in the B, molayi infected animals is bound to alter the permeability of the CNS
membrane, and cause disturbances in the concen!mtion of ~ g " and ca2'. The decreased
acetylcholinesterase activity in the present study may be due to altered lipid fluidity of the
CNS membrane because of peroxidation (Howard and Sawyer, 1980).
yGTP is an extrinsic membrane glycoprotein (Tate and Meister, 1976) widely
distributed in kidney, brain and liver. Brain endothelial cells are also rich yGTP and alkaline
phosphatase, which are responsible for transendothelial hansport and vascular permeability
(Vorbrodt el al', 1986). Tripathi er a/. (1994) reported a decrease in the yGTP and atkalie
phosphatase activity in the cerebral microcapillaries of mice infected with P. yoelii. In the
present study, the decrease in y-GTP and alkaline phosphatase due to B.mnlnyi infection
indicates membrane damage in the brain. The increased LPO (section 4.1.2.2) may be
responsible for the membrane damage of the brain cells. Moreover, the decrease in ahkine
phosphatase and y-GTP and acetylcholinesterase may increase disruption in blood brain
barrier permeability and lead to abnormal cerebral metabolism.
4.23.2 Membrane bound enzymes of RBCs.
Oxygen radicals cause more damage to red cells than to other cells, since they are
exposed to high oxygen concentrations in the lungs. The red cell membrane contains
unusually high concentration of vulnerable unsaturated fatty acids and lacks the cytochrome
respiratory pathway, which allows most of the oxygen in other cells to avoid sequential
acquisition of electrons (Suhail and Rizvi, 1990). Thus, reactive forms of oxygen are
generated at a higher rate in red cells and the balance between this stress and their
antioxidant defmces determines their life span (Carrel et al., 1975).
Mice red cells infected with P. vinckei had been reported to d9)age the membrane
calcium hansporl system due to the accumulation of rnalondialdehyde (Kayen ef al., 1983),
thereby increasing intracellular calcium. Tanabe et al. (1983) have shown reduced ca2'
ATPase and M ~ Z ' ATPase in P, chaubadi cells. They had also reported that the N a y
ATPase activity of the host cell membrane was 85% less than the normal. So the
permeability changed due to decreased ATPase, which would have resulted from the
membrane lipid changes in the RBC
The observed decrease in N ~ - K ' ATPase, ca2' ATPase and M~~ ATPase and
acetylcholinesterase activity in RBC in the present study indicates membrane damage. The
reduction in the activity of ATPases can be explained from the fact that they are lipid and
thiol dependent enzymes. and their decreased activity can be attributed to membrane damage
as reported above. Decrease in ca2* ATPase and M ~ ~ ' ATPase activity may be due to
peroxidation of the membrane lip~ds. The erythrocyte membrane is normally less permeable
to extracellular Ca2'. Enhanced host membrane permeability to Ca2+ is combined with
reduced activity of Ca2' ATPase, leading to an increase In intracellular ca2' (Sarkadi, 1980).
Acetylcholinesterase is an erythrocyte specific enzyme (Mitchell el al., 1965).
Elevated cholinesterase in mouse erythrocytes infected with P. chaubadi (Konigk and Mitsh,
1977), and P. berghei (Areekul and Cheerarnakara, 1987) had been reported. The decrease in
acetylcholinesterase in 5. malayi infected animals may be due to altered lipid fluidity of the
cell membrane (Howard and Sawyer. 1980).
4.233 Haematological parameters
Butteworth (1977) and Ogilvie et al. (1978) observed changes in the absolute count
of neutmphils. lymphocytes. monocytes and granulocytes in helminthic mfection. Choong
and Mak (1991) reported an increase in total count, differential counts and blood eosinophils
to k times at 3 weeks post infection upto 13 weeks in Presbytis cristata infected with 5.
Malayi. It further increased to five t ims upto 20 weeks, but no change in total leucocyte
count was observed. An increase in neutrophils was reported in Toxoplasma mfection
(Frcnkel. 1973). Rahmah el 01. (1994) reported that Toxoplarma gondi infection resulted in
the increase in neutrophils and cosinophils and a decrease in lymphocytes. Larsh (1967) and
Cypas ( 1972) reported infection of mice by Trichinella spimlir and Nematospiroides dubia
with i n d lymphocytes and decreased neutrophils. In P. v i v a and P. falcipamm
infection, decreased lymphocytes, increased monocytes and eosinophils had been observed
without significant changesin total leucocyte, neutrophil and platelet counts (Kurnaresan and
Selvam, 1991). Eosinophils and neutrophils play a significant role in host defense against the
parasites. In Onchocerciasis infections, eosinophils were shown to be the major participants
in adherence to 0. wlvulus (William er al., 1987). The cationic proteins of the eosinophil
damage target cells through membrane interaction, since the major basic protein as well as
eosinophil cationic proteins can disrupt cell membrane. Mackenzie (1980) had shown that
eosinophils eventually target mf and Greene er 01. (1981) had shown that neutrophils are
equally efficient in the same system.
In the present study the haernoglobin levels and RBC counts were not altered due to
the B. mnloyi mfection, while total WBC counts showed a decrease below nonnal values.
The significant increase in neutmphil and eosinophil counts and decrease in lymphocytes
confirm the above findings.
CHAPTER 4.3
CHANGES IN THE TESTES OF M. NATALENSIS INFECTED WITH
B. MALAY1
43.1 MATERIALS AND METHODS
4.3.1.1 Testosterone
Testosterone Radioimmunoassay was done using Testosterone Double Antibody kit
obtained from Diagnostics Products Corporation, Los Angeles, USA.
The basic principle of FUA involves competition between a radioactive and non-
radioactive antigen for a fixed number of antibody binding sites in a fixed time. The amount
of labeled analyte bound to the antibody (goat anti-rabbit gamma globulin) is inversely
proportional to the concentration of the analyte present in the sample. The antiserum is very
specific for testosterone with an extremely low cross-reactivity to other steroids.
Martomys rats were infected wth B. rnalayi as desnibed earlier. The testes was
excised after 0, 30, 60, 90, 120 days post inoculation, washed with phosphate buffered
saline. pH 7.4. homogenized and centrifuged at 2000 rpm for 20 minutes. The pellet was
discarded and the supernatant was used as sample for RIA.
Reagents
1. Testosterone antiserum
2. ('"1) Testosterone
3. Calibrators (conc. 5, 10,20,50, 100 & 200 pgtube)
4. Goat anti-rabbit gamma globulin
5. Polyethylene glycol
6. Borate buffer for testosterone
Tubes were labeled for calibrators, blank, maximum binding tube, total counts and
samples (tissue homogenate) in duplicate. Two hundred pl of the buffer was taken into the
blank tube and 100 pl ihto the maximum binding tube. Similarly 100 pi of the calibrators
and the sample was taken in the calibrator tubes and sample tubes. Then 100 p1 of the [I2' I]
testosterone was added into all the tubes, followed by 100 p1 of testosterone antiserum to all
the tubes except the blank tubes. The tubes were vortexed and incubated for a minimum of
60 minutes at R.T., which was followed by the addition of 100 pi of goat anti-rabbit gamma
globulin to all the tubes. The contents of the tubes were vortexed and incubated for I hr at
R.T. then 2.0 rnl of cold 4% PEG-saline solution was added and centrifuged at 2,000g for 20
minutes. The supernatant was decanted, the rim of each tube was blotted dry and counted in
a Gamma Counter for 1 minute. Calibration curve was prepared as indicated by the kit
procedure. The testosterone levels were expressed in nanornoles/mg protein.
43.1.2 7-GTP
1-GTP was assayed according to the method of Orlowski and Meister (1965) and
modified by Rosalki and Rau (1972) as described earlier.
43.13 LDH-X
The testes of M.naralemis infected with B.rnalayi were excised on different days of
post inoculation (0,30, 60, 90. 120). washed in chilled potassium phosphate buffer (0.1 M,
pH 8.3) and homogenised in the same buffer. The homogenate was centrifuged at 10,000g
for 30 minutes at 4°C and the supernatant was used for LDH-X isoenzyrne assay.
LDH-X isorymes were visualized on agar gel by following the method of Hanis and
Hopkinson ( 1978).
Reagents for staining
1. 0.05 M Tris-HCl buffer. pH 8.0 : 20 ml
2. Calcium l,actate (pentahydrate) : 100 mg (8 mM final concentration in reaction
mixture).
3. NAD
4. MTT : 5 mg in 1 ml water
5. Phenazine metho suiphate (PMS) : 2.5 rng in 0.5 ml water
6. Agar (approx., 2%) :20ml .
Electrophoresis conditions
1. Bridge buffer: 0.2 M phosphate buffer, pH 7.0
Gel buffer : 0.01 M phosphate buffer. pH 7.0
2. I2 Vlcm for 5 b, with cooling.
One percent agarose gel was prepared in 0.01 M phosphate buffer and fvted on a
horizontal electrophoretic system. About 10 p1 of each sample were added into individual
well along with the control sample. Whatman filter paper was used as the wick between the
bridge buffer and electrophoresis was carried out at constant current (I2 V) for 5 hrs. The
isoenzymes were visualised by activity staining as follows. The unfixed gel was rinsed with
cold his buffer (0.1 M, pH 8.3) and incubated for 10 minutes at 37C in dark in a staining
solution containing 200 p1 calcium lactate, 10 mg NAD, 5 mglml m, 2.5 md0.5 ml PMS
in 2% agar made upto 25 ml. The isoenzymes LDH 1-4 migrated anodally, while LDH-5
migrated cathodally. The LDH-X isoenzyme migrates between LDH-3 and LDH 4.
43.2. RESULTS
The effect of B. ma lay^ on testosterone, y -GTP and LDH-X of testes was studied and
the data are presented in Figs. 114 8~115. The testosterone levels were observed to increase
initially (upto 60 days) and decreased significantly w0.05) thereafter. And althrough the
study period. the activity of this hormone was always lower than the control.
y-GTP level increased significantly WO.05) in the infected group during the
parasite development than in control throughout the study period.
The LDH-X mzyme was visualized on agarose gel by activity staining (Platel). The
LDH-X bands in the infected animals were clearly visible on 30th day and faintly visible on
60th day but absenthot visible on 90th and 120th day, whereas. in control the enzyme bands
were clearly visible throughout the study period.
9 9
Fig.114: Effect of B, malayi on GTP testes of M. natalensis
Days of infection
- gamma GTP-N - gamma GTP-I
Fig.115: Effect of 8. malayi on Testosterone in testes of M. natalensis
104 0 30 60 90 1
Days of infection
Plate 1: Electrophoresis of LDH -X isoenzymes from testes of M. natalensis infected with B. malayi
L D H - I .
LDH - 2
LDH - 3
LDH - X LDH - 4
Lane 1 - Control (matured adult)
Lane 2 - 30 days post infection
Lane 3 - 60 days post infection
Lane 4 - 90 days post infection
Lane 5 - 120 days post infection
Lane 6 - Control (young adult)
433 DISCUSSION
Testosterone is a sensitive indicator of the reproductive function (Rikihisa et al.,
1984). The present study demonstrates that the testosterone production in the host was
decreased in the infected animals. Reduction in testosterone level may be due to degadation
or decreased testosterone output from the testes due to parasitic infection (Rikihisa et al.,
1985). Decreased number of leydig cells or their topological changes in distribution with
reference to blood and lymphatic vessels might cause decreased out put of testosterone in
testes. Rikihisa el al. (1985) reported a decrease in rat testosterone due to Taenia
taeniafonnis infection. A similar kind of inhibition is reported in Toxoplasma gondii
infection (Dias and Stahl, 1984).
y-GTP in the testes is primarily found in the sertolt cells of mammals and had been
used as a sertoli cell marker in rats (Hodgen and Sherins, 1973; Lu and Steinberger, 1977;
Fukuota et a/ . , 1990)). Hodgen and Sherins (1973) found an abrupt increase in y-GTP
activity in prepubertal rat testis which coincided with the cessation of sertoli cell division and
their maturation. Krueger er al. (1974) measured 1-GTP activity in testis of vitamin-A
deficient rats. The level of this enzyme ~ncreased during germinal cell depletion. The
observed increase in y-GTP In B.malayi infected animals suggests that the sertoli cell has
been damaged and germinal cells depleted. These adverse effects due to B. malayi infection
have been confirmed by the hstopathological studies (sec 4.4.2.1.3).
Among vertebrate species, somat~c cells often contain lactic dehydrogenase (LDH)
in an array of five isoenzymes. The unique LDH-X isoenzyme occurs in the mature testis of
mammals (Blackshaw, 1973; Linda and Mayeda, 1974; Taylor and Gutteridge, 1986). It is a
unique marker of the germinal cell rnaturatlon and the production of the sperms (Lalwani et
al., 19%). Hodgen (1983) show4 the coincidence of LDH-X with the histological
appearance of pachytene spematocytes. ltoh and Ozasa (1985) and Aganval el al. (1997)
reported impcurment of testicular function due to cadmium chloride administration to rats,
i.e., a marked duction in testicular LDH-X. The occurrence of LDH-X either in traces or
its absence in B. malayi infected animals observed in the present study may be due to the
reduced production of spermatocytes and sperms and this has been confirmed by the
histopathological studies'(section 4.4.2.1.3). And it may also be due to the decrease in the
testosterone levels and damage of sertoli cells.
CHAPTER 4.4
HISTOPATHOLOGICAL CHANGES IN MASTOMYS NATALENSIS INFECTED
WITH BRUGIA MALAY1 AND ITS RELATION TO DEC TREATMENT
4.4.1 MATERIALS AND METHODS
A group of 16 animals were maintained for 120 days, of wh~ch half sewed as
control and the other half were infected with B. malayi. Four animals each from the
control and infected group were administered with DEC (as described earlier) and
maintained for comparison purpose. Animals were euthanized using diethyl ether and
necropsied. Gross pathology was studied after systematic dissection. Representative
tissues from lungs, testes, heart, brain, liver, kidney and spleen were taken and fixed in
10% buffered formalin. Tissues were processed by routine paraffin embedding technique
and 5 micron thick sections were prepared and stained with hematoxylin and eosin and
examined under light microscope.
4.4.2 RESULTS
4.4.2.1 Studies after B. malayi infection
4.4.2.1.1 Lungs
The lungs of the control animals did not have any pathological change except for
the presence of few RBC's in the alveoli. In the infected animals, the lesions Included
mild exfoliative changes (Plate 2) in bronchiolar epithelium and moderate to severe
haemorrhages (Plate 3). ln such areas macrophages laden with haemosederin pigment
were also observed (Plate 4). Either sections or complete mf were seen in lumen of blood
vessels (Plates 5 & 6). perivascular space within the extravasited blood vessel, in the
connective tissue (Plate 6) and beneath the pluera. In some of the areas, mf was
surrounded by mononuclear infiltratory cells. Presence of giant cells suggests the
chronicity of the lesions (Plate 8). In a few infected animals, adult parasites were seen in
pockets (Plate 9) and out of them a few wen ncognised as adult female due to the
presence of mf in them. (Plate 10).
Plate2 Section of Lung of infected group showing exfoliation of bronchiola
epithelial cells. (Haematoxylin and Eosin 400X)
Plates: Section of Lung of infected group showing severe hemorrhages. (Haematoxylin
and Eosin 400X)
Plate Q: Section of Lung of infected group showing haemosiderosis in the areas of
h e m o d g e (Haematoxylin and Eosin 400X)
Pla tes Section of Lung of infected group showing microfilana in lumen of blood
vessel (Haematoxylin and Eosin I OOX).
Plate 6 Section of Lung of infected group showing microfilaria across the blood vascular
wall (Haematoxylin and Eosin 400X).
Plate? Section of Lung of infected group showing a complete coiled microfilaria
extravascularly (Haematoxylin and Eosin IOOOX).
.
Platc 2 Plate 3
Plate 4 Plate 5
Plate 6 Plate 7
Plate$: Section of Lung of infected group showing foreign body giant cells indicating
the chronicity of lesions (Haematoxylin and Eosin 1000X).
Plateg: Section of Lung of infected group showing adult parasite (Haematoxylin and
b i n IOOX).
Platew Section of Lung of mfected group showing cross sections of adult female parasite
with microfilaria (Haematoxylin and Eosin IOOOX).
Plate fi : Secnon of Heart of infected p u p showing moderate infiltration of
mononuclear cells in epicardium. (Haematoxylin and b i n 400X).
Plate 13 Section of H e m of u L f d group showing microfilaria in myocardium
(Haanatoxylin and b i n 1000X).
Plate \k Sectron of Heart of infected group showing microtilaria and hemorrhage in
myoca&rn (Haematoxylin and Eosin 1000X).
Platc X Plate 9
Plate 10 Plate 11
Plate 12 Plate 13
4.4.2.1.2 Heart
The heart of the control animals did not show any other significant change.
However, in the infected animals, a few mononuclear inflammatory cells were seen in the
connective tissue and in the myocardium around the parasite. In the epicardium,
moderate to severe infiltration of mononuclear cells was observed (Plate I I). Some of the
muscle fibres in the myocardium had a few fat vacuoles. Microfilariae were seen in the
myocardium (Plate 12), in the capillaries and in interstitial connective tissues. Presence
of some erythrocytes in the vicinity of the mf indicated haemorrhage (Plate 13).
4.4.2.1.3 Testes
The testes of the control group had seminiferous tubules with normal
spermatogenesis. The lumina of the tubules were filled with many sperms (Plate 14). The
interstitial tissue had normal number of leydig cells. In this group, no circulatory or
degenerative change was noticed.
In the infected animals, though some of the tubules appeared normal, many of
them did not have multilayered spermatogenic cells. The lumina contained only a few
developed sperms (Plate IS). When compared to the control, the testicular damage was
marked. A few tubules showed initial stages of calcification. In some areas, the
interstitial cell of leydig was normal, but in the areas of tubular damage, these cells also
became degenerated and atrophied.
4.4.2.1.4 Liver
The liver of the control animals did not have any significant change. In the
infected group, vacuolation of hepatocytes was more pronounced and in addition, some
of the hcpatocytes showed fatty changes and necrosis (Plate 16). Portal veins showed
hyperaemia. Nuclear hyperchromacia and presence of twin nuclei in some hepatocytes
suggested an ongoing regenerative process (Plate 17). A few small foci of mononuclear
cellular infiltration particularly in the areas of hepatic degeneration were also recorded
(Platel8). Microfilaria was mainly in the sinusoids (Plate 19).
Plate w: Section of Testes of normal group showing seminifmus tubules with
normal spermatogenesis (Haematoxylin and Eosin 400X).
Plate 19 Section of Testes of infected group showing moderate reduction in
spermatogenesis indicating testicular degeneration (Haematoxy tin
and Eosin 400x1.
Plate Y: W o n of Liver of infected group showing hydropic degeneration of fatty
changes in the perilobular areas (Hamatoxylin and Eosin 100X).
Plate IT Section of Liver of infected group showing hyperchromatic binucleated
hepatocytes indicating regenerative p r w m (Haematoxylin and
=n 400X).
Plate 18: Section of Liver of infected group showing hepatic degeneration with
moderate mononuclear cellular infiltration (Haematoxylin and Eosin 2SOX).
Plate 19: Section of Liver of infected group showing microfilaria in hepatic sinusoids
(Hacmatoxylin and Eosin 400X).
Platc 14 Plate 15
Platc I6 Plate 17
Plate 18 Plate 19
4.4.2.1.5 Brain
The control animals did not have any significant pathological changes.Mild
congestion of the meningeal blood vessel was noticed in the infected brain (Plate 20).
Mild neuronal degeneration and a few areas showing satellites and neurophagia were also
seen. Around some of the blood vessels of brain tissue of infected animals, widening of
Virchow- Robin's perivascular space was a feature (Plate 2 1. Numerous sections of mf
were seen in the lumen of blood vessels and in the brain tissue (Plates 22 & 23). Around
a few mf, the infiltration by glia cells was also recorded (Plate 24).
4.4.2.1.6. Kidney
In the control group, kidneys did not show any other significant histological
change. However, in the infected animals, the renal tubules showed moderate to severe
vacuolar degeneration of the tubular epithelium (Plate 25) . In a few tubules, tubular
necrosis leading to formation of hyaline casts was noticed. The hyaline casts were more
in the tubules of the medullary region (Plate 26). In the renal parenchyma, some areas
showed mild haemorrhage. The subcapsular space was widened in some places and
showed mf (Plate 27). Sections of mf were also noticed in the glomerular tuft (Plate 28),
interstitium (Plate 29) and in some large renal blood vessels.
4.4.2.1.7 Spleen
In the control animals, spleen did not show any significant pathological change.
The amount of hemosiderin pigment was in normal quantity. However, the infected group
showed lymphoid hyperplasia (Plate 30) and presence of giant cells (Plate 31). No mf
was observed in the section of spleenic tissue.
Plate a Section of Brain of infected p u p showing congestion of meningeal blood
vessel (Haernatoxylin and Eosin IOOX).
Plate 3: Section of Brain of infected p u p showing widening of Varsho-Robins
perivascular space (Haernatoxylin and Eosin 400X).
Plate a Section of Brain of infected group showing microfilaria in the blood vessel
(Haematoxylin and Eosin 400X).
Plate fa: Section of Brain of infected group showing microfilaria (Haematoxylin and
Eosin 400X).
Plate a: Section of Brain of infected p u p showing infilhation of glial cells around
microfilaria (Haematoxylin and Eosin 400X).
Plate 15 W o n of hdncy of ~nfected group showing severe vacuolar degeneration
in the tubular epithelial cells (Haernatoxylin and Eosin 250X).
Plate ICt Section of Kidney of infected group showing hyaline casts in the lumen of
nedu1lary tubules (Haematoxylin and Eosin ZSOX).
Plate 17 Section of Kidney of infected group showing nicrofilaria in subscapsular
tissue (Haematoxylin and E&i I W X ) .
Plate a Section of Kidney of infected group showing microfilaria in glonemlar tufts
(Haematoxylin and Eosin 1000X).
Plate P W o n of Kidney of infected group showing nicrofilaria in renal interstitial
tissue (Harmatoxylin and Eosin IOOOX).
Plates )Om of Spleen of infected p u p showing lymphoid hypcrplacia
(Haanatoxylin and W i n 250X).
Plate31: Sea~on of Spleen of infected group showing giant cells with Langhan's type
of appearme (Haematoxylin and Eosin 400X).
I'iatc 26 Plate 2:
4.4.2.2 Histopathological changes in relation to DEC treatment
All the organs of the infected DEC treated animals showed similar histological
changes like that of the infected untreated animals. However, the testicular tissue showed
severe degeneration and the seminiferous tubules were completely devoid of
spermatogenic cells and spermatozoa (Plate 32). They contained only fibrous material. In
some areas, moderate to severe infiltration of the lymphocytes, plasma cells and
macrophages were recorded. Adult female parasite sections were present in pockets
(Plate 33). In some sections calcified adult parasites were observed. Mild connective
tissue proliferation was present around the parasite in some sections.
4.4.3. DISCUSSION
Vincent et al. (1976) studied the chronological development of pulmonary
pathology associated with B. malayi, B. pahangi and B. patei in Meriones unguiculatus,
which caused inflammatory reactions. Pulmonary granulomas were observed during the
final molt, followed by involution and formation of residual vascular lesions and some
were seen during sexual maturity of the worms. Obstructive endarteritis and chronic
interstitial inflammation with degenerating mf were also observed. Malone et al. , (1976)
studied the histopathological lesions in the lymphatic system and other major organs of
hamsters infected with B. pahangi. Cellular infiltration of plasma cells and eosinophil,
obstruction of pulmonary arteries and obstructive granulomatous lymphangitis were
observed. Live and dead worms were found in testicular parenchyma. Accumulation of
eosinophils, large mononuclear cells and plasma cells were seen in interstitial tissues
between superfacial seminiferous tubules. Heavy accumulation of hemosiderin and giant
cells were also observed in the lung. Degenerative or necrotic hepatocytes occurred In the
liver. Schacher and Sahyoun (1967) reported pathologic changes due to B. pahangi in
experimentally infected cats and dogs. Hyperplasia of lymph follicles and reticular cells
of the nodal stroma had been reported (Schacher and Sahyoun, 1967; Mak, 1983).
Destruction of mf had-been observed in the spleen, which caused acute and chronic
inflammatory reaction in patients infected with B. malayi (Mak, 1983). Large number of
lesions were observed in liver, lungs and spleen of ferret infected with B, malayi and B.
Plate 31: Section of Testes of infected DEC treated group showing severe testicular
degeneration and C.S. of adult parasite (Haernatoxylin and Eosin IOOX).
' Plate Section of Testes of infected DEC treated group showing sections of adult
parasite (Haematoxylin and Eosin IOOX).
Plate 3 2
pahangi and no lesions in the kidneys (Crandell et al., 1982). Case et al. (1991) reported
eosinophilic abcesses and epithelioid and giant cell granulomas with fragmented worms
in various stages of disintegration in kidney, spleen, liver, lung, pulmonary blood vessel
and lymphatics of ferrets infected with B. malayi. Endothelial hyperplasia, blood vessel
obliteration with marked perivascular infiltration of lymphocytes, plasma cells,
eosinophils and numerous large macrophages laden with a coarse golden brown pigment
was also reported.
The pathological changes observed in the present study in various organs were of
inflammatory and degenerative nature suggesting tissue injury and body's response.
Since the mf circulated mainly in the blood vascular system and acted as parasitic emboli,
they might have got lost in smaller blood vessels, such as arterioles and capillaries of
various organs. This might have resulted in ischemia, tissue hypoxia and subsequent cell
injury seen in the form of various retrogressive changes. Toxic metabolic products of the
mf and adult parasites and hepatic damage leading to decreased detoxification capacity
can be considered as added factors in causation of degenerative changes in all the organs.
The inflammatory changes in various organs might be in response to the mf and adult
parasites as well as the necrosed tissues, which acted as foreign body. This is in
agreement with earlier reports by Schacher and Sahyoun (1967) and Malone el al.
(1976).
The adult parasite in the testes caused significant inflammatory changes, which
led to severe damage to spermatogenesis. In an earlier study adult worms had been
reported in testes (Malone ef al., 1976), but no significant pathological changes were
seen. In the present study the testicular damage after DEC treatment was more
pronounced. Since the DEC treatment was given after the development of parasites, they
got killed, calcified and elicited a foreign body reaction. Hence the changes became more
pronounced and testicular damage could not be reverted back. Further studies will be
required to understand the action of DEC in relation to stage of infection and possible
pathological outcome.
The mf could not be seen in spleen in B.rnalayi infected animals as reported
earlier. This may be because it is an important organ of immune system of the body. The
fact is substantiated by mild! lymphoid hyperplacia and presence of giant cells observed
in spleen of infected animals. This is in agreement with earlier reports by Duke (1960)
and Mak et al. (1984), where spleen was unaffected by 5, malayi infection. Thus the
morphological changes seen in the spleen would be dependent on the state of the host's
patho-immunological responses. To find the specific cells of the immune system
responsible for killing of mf and the irreversible testicular damage may be another
interesting field for further investigation.