Comparative Parasitology 67(2) 2000 - Peru State College

is. Using histochemical methods our group demonstrated
NOS in basophilically transformed
muscle fibers in T. spiralis—infected mice (Hadas
et al., 1999). In a separate paper we reported on
the participation of ROS in the biochemical protective
mechanisms in host muscle infected with
T. spiralis larvae (Wandurska-Nowak et al.,
1998). In the same paper we demonstrated that
administration of the glucocorticoid methylprednisolone
had a profound effect on the activity of
antioxidant enzymes that were examined (superoxide
dismutase [SOD] and peroxidase). According
to Connors and Moncada (1991), glucocorticoid
also inhibits iNOS.
The initiation of research on the participation
of iNOS in biochemical defense mechanisms of
the host in T. spiralis infection was also important
from the point of view of its possible participation
in the mechanism of uncoupling of oxidative
phosphorylation, which can be observed
in the mitochondria of tissue infected with helminths
(Michejda and Boczori, 1972; Van den
Bosche et al., 1980; Boczori and Bier, 1986;
Ruble et al., 1989). It was shown that the expected
temporal correlation between the increase
in the activity of SOD and peroxidase and the
peaks in trichinellosis phosphorylation uncoupling
did not occur (Wandurska-Nowak et al.,
1998).
The objectives of the present investigation
were to determine 1) quantitative changes in the
activity of iNOS in muscles from hosts infected
with T. spiralis or Trichinella pseudospiralis
Garkavi, 1976, and 2) if glucocorticoid prevents
changes in the quantity of NO generated in infected
tissues.
Materials and Methods
Experimental tissue consisted of muscles removed
from uninfected mice (2-mo-old female mice, strain
BALB/C) and from mice infected per os with 700-800
infective larvae of either T. spiralis (strain MSUS/PO/
60/ISS3) or T. pseudospiralis (strain MPRO/US/72/
ISS13). The infective larvae obtained after pepsin-HCl
digestion after about 2 hr for T. spiralis larvae and
about 1-1.5 hr for T. pseudospiralis were administered
per os to mice anesthetized with ether. The mice were
killed by decapitation. The amount of larvae per 1 g
of muscle tissue obtained after pepsin-HCl digestion
at 6-8 wk post-infection (p.i.) were 10,000-12,000 and
5,000 for T. spiralis and T. pseudospiralis, respectiveiy.
Mice were bred and housed in the animal laboratory,
which ensured approximately constant temperature,
humidity, and ad libitum access to LMS Labofeed B
BOCZON AND WARGIN—iNOS IN MOUSE MUSCLE 231
(Feed and Concentrates Production Plant) granulated
food and water.
Only 1 group of animals infected with T. spiralis
larvae was treated with methylprednisolone (Depomedrol
[Jelfa, Poland], a drug with prolonged action)
administered on day 7 p.i. by subcutaneous injection
at a dose of 20 mg/kg of body weight. Quadriceps
muscles from hind legs were removed and homogenized
for 15 to 30 sec in a sucrose medium of the
following content (in final concentration): 0.25 M sucrose,
0.002 M EGTA, 0.01 M Tris HC1 buffer (pH
7.3), and 20 jxl heparin with a concentration of 500
units/g per 10 ml medium. The homogenate was centrifuged
for 10 min at 4,500 rpm, and the resulting
supernatant was centrifuged for 12 min at 15,000 rpm.
The activity of iNOS was measured in the latter supernatant
spectrophotometrically by Green's method as
modified by Lepoivre (Lepoivre et al., 1989), using the
following solutions: A) Griess' reagent containing
0.5% sulphanilamide dissolved in 1 N HC1 and 0.15%
/V-(l-napthyl) ethylendiamine mixed in a ratio of 1:1
and B) consisting of (in final concentrations) 40 mM
Tris HC1 buffer (pH 8.0), 2 mM NADPH, and 7 mM
arginine. Enzyme activity was measured in 140 JJL! of
supernatant after 30 min of incubation (to induce the
enzyme activity) at 1-wk intervals at a wavelength of
X = 540 nm in a cuvette containing 1,200 u,l of solution
A and 100 u.1 of solution B. In some pilot experiments
1.5 mM CaCl2 was added. Absorption readings
were taken after a 30-min incubation period at a
temperature of 24 °C, and NO concentration was determined
using a NaNO2 standard curve. Protein was
measured applying Lowry's method (Lowry et al.,
1951).
The measurements were carried out in 4 groups of
animals: for T. spiralis-infected mice (I+NaCl), T.
spiral is-infected mice under treatment (I+D), and also
for 2 control groups (C+NaCl and C+D). Both infected
and untreated mice (I+NaCl) and those from
the respective control group (C + NaCl) were given intramuscular
injections of 0.9% NaCl. Activity measured
in the respective control groups was taken as
100% for the calculation of percentage changes in such
activity during T. spiralis infection and treatment.
Analysis of variance or the Mann—Whitney test was
used for statistical comparison between groups; P <
0.01 (very significant) or