When the primary tumor reached a size of 50-100 mm
3
, 40 mice were grouped into 4 groups 10 mice each. Group 1 received normal saline EAC-bearing
control, 5 mgkg. Group 2 received BLM 5 mgkg from 0.1 solution I.P every other day for 10 days. Group 3
received paracetamol 200 mgkg, orally, Hunskaar et al., 1985 for 10 consecutive days. Group 4 received BLM
and paracetamol. Tumor size before day 0, five days after tumor inoculation and after treatment day 10,
fifteen days after tumor inoculation was measured. Antitumor activity was calculated by the determination
of T change of tumor size in the treatment group and C change of tumor size in the control. The degree of
tumor growth inhibition can be obtained from TC X 100 Schirner et al., 1998.
2.4.3. Lung toxicity
Rats were grouped into 4 groups of 8 rats each. Group 1 received normal saline 2.5 mgkg. Group 2
received BLM mgkg from 0.5 solution I.P. three timesweek for 4 weeks Daba et al., 2002. Group 3
received paracetamol 150 mgkg, orally, Chattopadhyay et al., 2002 daily for 4 weeks [13]. Group 4 received
BLM and paracetamol. Twenty-four hours after the last dose of the specific treatment, all rats were weighed and
then sacrificed by cervical dislocation. The pulmonary arteries, trachea and lungs were rapidly excised.
The vasoconstrictor effect of 5-HT on the pulmonary artery and the contractile response to ACh on
trachea were tested. The percent survival of rats was also recorded at the end of treatment after 4 weeks.
Total lungs were weighed for determination of lungbody weight ratio. Sections of the isolated lungs,
each weighing 100 mg, were then used for estimating collagen contents, while the other biochemical
parameters
Lipid peroxide,
nitratenitrite level,
superoxide dismutase activity and GSH were assessed in the lung homogenates. All died animals in the survival
test were replaced to complete 8 ratseach group for other tests.
2.4.4. Preparation of pulmonary arterial rings
Segments of the main pulmonary artery were rapidly placed in warm physiological salt solution PSS
Kreb’s enseleit and dissected free of surrounding tissue. The composition of PSS in mmolL was NaCl 118;
KCl 4.7; CaCl
2
2.5; KH
2
PO
4
1.2; MgSO
4
.7H
2
O 1.2; NaHCO
3
25 and glucose 10; pH 7.4 Blom-Muilwijk et al., 1988. The
pulmonary artery was trimmed and cut into rings about 2 mm in length. The rings were mounted horizontally
between a clamp and a force transducer in an organ bath filled with 50 ml of the PSS. The PSS was kept at
37
o
C and continuously bubbled with a mixture of 95 O
2
and 5 CO
2
. The suspended pulmonary arterial rings were allowed to equilibrate for 30 min under a resting
load of 3 g El-Kashef, 1996. The isometric tension was monitored by means of
a displacement transducer model 50-7905, Harvard Apparatus LTD., South Natick, MA, USA connected to a
two-channel physiograph recorder model 50-8622, Harvard Apparatus LTD., USA. The contractile responses
of the pulmonary arterial rings of each group of animals to different concentrations of 5-HT ranging from 10
-7
to 10
-5
M were recorded. The vascular responsiveness of the tissues to 5-HT was calculated after weighing the rings as
g tensiong tissue.
2.4.5. Preparation of trachea
The trachea was rapidly removed, placed in Kreb’s
Henseleit solution gassed with 95 O
2
5 CO
2
and kept at 32
o
C. The Zig-Zag tracheal strips Emmerson and Mackay, 1979 were then prepared. The tracheal tension
was set at 1 g and a stabilization period of 1 h, was allowed before treatment with drugs. The contractile
responses of the trachea of each group of animals to different concentrations of ACh ranging from 10
-6
to 10
-4
M were recorded. The trachea was weighed and the responsiveness to ACh was calculated as g tensiong
tissue.
2.4.6. Biochemical measurements 2.4.6.1 Determination of collagen
The collagen content in lung was assayed
indirectly as its hydrolytic product, hydroxyproline, HP Woessner, 1961. The absorbance was read at 557 nm
using colorimeter WPA colourwave Model CO 7500, Cambridge, England and the concentrations of collagen
were calculated as mg HPg wet tissue.
2.4.6.2 Preparation of lung homogenate
The isolated lungs were rinsed in chilled 1.15 KCl pH 7.4 and weighed quickly. Subsequently, the
lungbody weight
ratio was
determined. Homogenization was carried out in ice-cold KCl 1.15, pH
7.4 to yield 10 wv tissue homogenates Daba et al., 2002 and the following biochemical parameters were
assessed.
2.4.6.3 Determination of Lipid peroxidation LP
The level of LP in the lung was estimated as thiobarbituric acid reactive substances Ohkawa et al.,
1979. The absorbance was determined at 532 nm spectrophotometerically and the concentrations were
expressed as nmolg wet tissue.
2.4.6.4 Determination of reduced glutathione GSH
The level of acid-soluble thiols, mainly GSH, in the lung was assayed colourmetrically, based on its reaction
with Ellman’s reagent 1959 . The absorbance was measured at 412 nm and the concentrations were
expressed molg wet tissue.
2.4.6.5 Determination of superoxide dismutase SOD
The enzymatic activity of SOD was assessed according to Marklund 1985. SOD activity was
expressed as Ug wet tissue. One unit of SOD activity is defined as the amount of the enzyme causing 50
inhibition of auto-oxidation of pyrogallol.
2.4.6.6 Determination of nitric oxide NO
The total amount of NO produced in the lung was indirectly estimated as the main metabolites, nitrate and
82
Control BLM
P BLMP
25 50
75 100
S u
rv iv
a l
nitrite. After the conversion of nitrate to nitrite by enzyme nitrate reductase in the presence of the cofactor
NADPH, NO was collectively determined as nitrite by the Griess reaction. In acid solution, nitrite is converted to
nitrous acid HNO
2
which diazotizes sulfanilamide. This sulfanilamide-diazonium salt then reacts with N- 1-
Naphthyl-ethylenediamine dihydrochloride NEDA to produce a chromophore which is measured at 540 nm
and the concentrations of NO were calculated as mol of nitrite and nitrateg wet tissue Tracy et al., 1995.
2.5
Statistical analysis Data are expressed as mean S.E.M. Significance
was calculated at p0.05. Statistical analysis was carried out using one way analysis of variance ANOVA
followed by Tukey-Kramer multiple comparisons test , in addition to linear regression analysis for the best fitting
line of all standard points Daniel, 1991. Also paired Stud
ent’s t-test was used as a test of significance for comparison between two arithmetic means of the same
subject before and after treatment Daniel, 1991. Chi- square test was used for comparison of two proportions
Daniel, 1991. Statistical calculations were carried out using Instat-2 computer program GraphPad Software
Inc. V2.04, San Diego, CA, USA.
3. Results
