Does N-Acetylcysteine Have An Effect On Acetylcholine-Induced Contractions and Histopathological Changes On Isolated Rat Ileum?
Does N-Acetylcysteine Have An Effect On Acetylcholine-Induced Contractions and Histopathological Changes On Isolated Rat Ileum?
Does N-Acetylcysteine Have An Effect On Acetylcholine-Induced Contractions and Histopathological Changes On Isolated Rat Ileum?
Emine Koc, PhD, Sema A. Yavuzer, MD, Belgin Can, MD, Banu Ocakcıoglu, MD, Ahmet Ergun, MD, Yuksel Saran, Dt, PhD.
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Does NAC have effect on isolated rat ileum ... Koc et al
Two experimental groups were designed. The first glycol-bis (Beta-aminoethyl-ether)-N, N’-tetraacetic
group, the control group (n=7), had only saline (0.9% acid (EGTA) 3 mM and equimolar NaCl were added
NaCl) added to their water. The second group, the to the tyrode solutions instead of CaCl2. The ACh-
experimental group (n=7) was administrated NAC induced contraction amplitude was measured in mm
(Sigma Chem Co.) 0.5 g/Kg/day orally by adding to from recorded traces, and the calibration was made as
their water for 7 days. At the end of the experimental 1 g per 10 mm. The ileal tissue was examined using light
periods, under pentobarbital anesthesia (35 mg/Kg and electron microscopic technics for histopathological
intraperitoneal) the abdomens were opened and one changes. In the light microscopy studies, the ileal
cm pieces of terminal ileum segments were removed tissue was fixed in 10% formaldehyde, dehydrated in
rapidly for isolated tissue bath experiments, and one alcohol, and embedded in paraffin. Sections were cut
cm pieces of ileum were removed for histopathological in 5-6 microns and stained with Hematoxylin-Eosin
experiments. To measure the isometric contraction of (H-E). For electron microscopic examination, the
ileum, tissue segments were suspended in an isolated ileal tissue was fixed at 40C for a period of 1-2 hours
tissue bath containing 20 ml standard tyrode solution in 2.4% phosphate buffered glutaraldehyde solution,
(Tyrode solution in mM: sodium chloride (NaCl) 137, and postfixed in 1% osmium tetroxide. After alcohol
potassium chloride 2.7, magnesium chloride 1.05, dehydration, the samples were embedded in Araldite
Calcium Chloride (CaCl2) 1.8, sodium dihydrogen 6005. Sections were cut on a Leica Ultracut R with a
phosphate 0.42, sodium bicarbonate 11.90, glucose glass knife, stained with uranyl acetate, and lead citrate,
5.5) and bubbled with 95% oxygen, 5% Carbon and were viewed on a LEO 906 E transmission electron
Dioxide mixture at 370C, pH adjusted at 7.4. To microscope.
record tension, the samples were mounted vertically Statistical analysis was performed by Mann-Whitney-
between a fixed holder and a force transducer (Ugo U and Wilcoxon tests where appropriate, using SPSS
Basile isometric transducer No 7003) and the tissues software. The results were expressed as means ± SD.
were brought into equilibrium in 30 minutes under an Statistical significance was set for p<0.01 or p<0.05.
optimal resting tension of 0.3 g. The isometric tension
was recorded by an Ugo Basile no: 7050 recorders. Results. The ACh-induced contraction responses
Contraction of ileum segments was induced by adding of the experimental group and the control group both
ACh 2.7 x 10-7 M into the bath solutions. These in the standard tyrode and calcium-free tyrode group
procedures were repeated in the calcium-free medium. are summarized in Figure 1. The histopathological
When calcium-free solutions were used, 2 Methylene- changes observed by both light microscope and electron
Figure 1 - The changes of contraction amplitude as millimeters measured in standard and calcium-free tyrode solution in
control group and N-acetylcysteine (NAC) administrated group. Data are given as mean ± SD, P-significant
level for the changes experimental period, *p<0.05. **p<0.01, n=number of animals. The calibration is 1 g per
10 mm in our setup.
free SH group, and cysteine (which has disulfide bonds) decreased significantly in the control and experimental
has no radical scavenging activity, therefore the free SH groups (p<0.05, p<0.01). This finding is not interesting in
group might play a significant role in protecting the terms of the control group because of decreased calcium
cells from oxidant-induced cytotoxicity. Thus, NAC entry in calcium-free medium. However, contractile
might directly or indirectly, via its metabolites, act as a response was extremely reduced in the experimental and
scavenger of ROS.16 control groups in calcium-free medium. Therefore, we
We showed that NAC decreased the ACh-induced thought that NAC would act as a toxic substance with
ileal contraction, and therefore the ileal contractility. these doses, and these effects would cause a disturbed
When we designed the study, our hypothesis was energy metabolism as we showed in the histological
that NAC may increase the ileal contractility by its data. N-acetylcysteine seems to be able to protect
antioxidant effects. It was interesting to find decreased against apoptosis through mechanisms that maintain
contractility. So, therefore, we thought that the dosage mitochondrial integrity.25 Our histological findings
of NAC that we used was not enough to increase showed that, when 0.5 g/kg/day NAC was administrated,
GSH or, as Meister’s data indicates, it may be toxic mitochondria were swollen and cristae were disturbed.
at these doses.17 However, there are some reports that In contrast with our present results, previous results
are compatible with our findings. Lida et al18 thought showed that NAC administration had beneficial effects
that, in glioblastoma and glioma cell lines, intracellular on synaptic mitochondria, at least partially by its direct
GSH concentration was not changed by NAC; thus, antioxidant action. The NAC would act potentially on
the effect of NAC may depend on its radical scavenging mitochondrial oxidative phosphorylation complexes by
effect instead of its GSH modulating effect. Also, Alton possibly protecting oxidative damage by maintaining
Meister reported that administration of cysteine is not the protein sulfhydryl groups.26,27 The NAC could
an ideal therapy because it may be followed by toxic reduce the lipid peroxides in mitochondrial membrane
reactions.17 In addition, Kharazmi et al19 showed that that can impair mitochondrial complex activities.28,29 In
high concentrations of NAC has a strong cytotoxic contrast with these studies showing a beneficial effect of
effect on neutrophils. Vina et al20 demonstrated that NAC on mitochondria,26-29 our histological data show a
intraperitoneal injection of cysteine or NAC causes swollen mitochondria and disturbed cristae, in which the
depletion of GSH in rat brain.20 inner mitochondrial membrane (cristae) is responsible
Our findings suggested that 0.5 g/kg/day NAC for the mitochondrial production of ATP in aerobic
administration (although we cannot determine the ROS metabolism. The possibility that the dose of NAC in
activity) may cause an increase in ROS production, which the present experiment was insufficient to modify
affects ACh-induced contraction. Our histopathological complex specific activities on cells that would increase
findings supported the physiological data. As shown in GSH in cytosol and complex antioxidant activities in
Figures 3 & 4, there are large numbers of mitochondria, mitochondria exists. Our results agree with the report
and some of the mitochondria appeared slightly swollen, that, some antioxidants, cysteine, NAC, and GSH,
and had disturbed cristae. Mitochondria degeneration significantly reduced radical intensity and cytotoxic
may be due to the presence of abnormal collections activity and these antioxidants induced cytotoxicity via
of mitochondria since high-energy requirements of their pro-oxidants action.8
contracting muscle cell cannot be metabolized. It was In conclusion, we showed that NAC decreased the
shown that mitochondrial lesions can diverse cellular ACh-induced contractions on isolated ileal strips by yet
bioenergetic capacity and also increase the generation of undefined mechanisms. In view of our data, and since no
ROS.21 Martensson et al15 found that chronic depletion other study seems to have reported the effect of NAC on
of mucosal GSH in mice causes severe degeneration of isolated ileal contractility, we can say that, although we did
the epithelial cells of the jejenum and colon. Lash et not examine the ileal levels of ROS or GSH, the dietary
al22 demonstrated that GSH concentration diminishes supplementation of NAC decreases ileal contractility,
60% in isolated intestinal epithelial cells after one hour probably by increasing ROS activity in mitochondria.
of incubation in reduction free of GSH or precursor
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