2Marmara University Vocational School of Health Related Professions, Istanbul, Türkiye
3Marmara University School of Medicine, Histology & Embryology, Istanbul, Türkiye
4Marmara University School of Medicine, Physiology, Istanbul, Türkiye DOI : 10.12991/201014462
Summary
Bu çalışmada nikotinin ürogenital sistem üzerinde oluşturduğu oksidan hasara karşı resveratrolün koruyucu etkileri biyokimyasal, histolojik ve fonksiyonel olarak araştırılması amaçlanmıştır. Wistar Albino sıçanlara 28 gün boyunca 0.6 mg/kg dozunda nikotin hidrojen bitartarat veya serum fizyolojik intraperitoneal olarak enjekte edildi. Bu hayvanlara aynı zamanda resveratrol (10 mg/kg) oral yolla uygulandı. Dekapitasyon sonrası mesane, korpus kavernoz ve böbrek dokuları çıkarıldı. Kavernoz ve mesane dokularında in vitro kontraktilite çalışmaları yapılırken böbrek dokusu da dahil her üç dokudan alınan örnekler malondialdehit (MDA), glutatyon, luminol ve lusigenin kemilüminesans (KL) düzeyleri ölçümleri için -80 °C'de saklandı. Dokular histolojik olarak da incelendi. Kronik nikotin uygulamasının böbrek, mesane ve korpus kavernozum dokusunda GSH düzeylerinde neden olduğu anlamlı azalma, MDA, luminol– lusigenin KL düzeylerinde neden olduğu anlamlı artışlar histolojik olarak da belirlenen oksidatif hasarı gösterdi. Nikotin uygulamasının neden olduğu serumda kan üre azotu, kreatinin, proinflamatuar sitokinler (TNF-α ve IL-1β), laktat dehidrojenaz aktivitesi, oksidatif DNA hasarı (8-OHdG) artışları ve antioksidan kapasitedeki azalma resveratrol tedavisi ile geri çevrildi. Ayrıca kronik nikotin uygulamasının mesane ve korpus kavernozumda neden olduğu kasılma ve gevşeme yanıtlarındaki değişiklikler yine resveratrol tedavisi ile düzeldi. Resveratrol tedavisi tüm dokularda endojen GSH düzeylerini iyileştrerek oksidan hasar parametrelerini azalttı. Bu sonuçlar resveratrolün muhtemelen antioksidan etkisi ile kronik nikotinin böbrek, mesane ve korpus kavernoz dokularındaki zararlı etkilerini karşı koruyucu olduğunu göstermektedir.Introduction
Tobacco smoking is one of the leading causes of death in both the developed and the developing countries[1]. Among numerous harmful substances in tobacco, the primary addictive substance is nicotine. Nicotine, a water-soluble alkaloid which may be acquired through active and passive smoking, is rapidly absorbed through the respiratory tract, gastrointestinal tract, skin and mucous membranes and it is mainly metabolized in the liver[2]. Experiments have shown that chronic administration of nicotine causes increased lipid peroxidation products in the serum and various tissues of rats[3,4]. Oxidative cellular damage due to nicotine-induced chronic inflammation is associated with generation of reactive oxygen species in the periphery and central nervous system resulting in an imbalance in the cellular oxidant-antioxidant system[5-8].Resveratrol is a potent member of the class of natural, plant-derived chemicals known as polyphenols, which are synthesized by a wide diversity of plants, such grapes, raspberries, mulberries, pistachios and peanuts, in response to stress, injury, ultraviolet irradiation and fungal infection as part of their defense mechanism[9]. Polyphenols have a variety of biological functions, including antioxidant, anti-inflammatory, and anticancer effects[10-13]. Resveratrol protects the cardiovascular system by exerting a positive effect on both the progression and regression of atherosclerosis, by inhibiting low-density lipoprotein (LDL) oxidation and blood platelet aggregation. Experimental studies have shown that this natural product possesses anti-inflammatory properties and inhibits the growth of some tumors[14-17].
Cigarette smoking in males has been implicated as a cause of decreased sperm numbers and an increased frequency of abnormal sperm morphology as well as a decrease in sexual performance[18]. Clinical and basic science studies provide strong indirect evidence that smoking may affect penile erection by the impairment of endothelium-dependent smooth muscle relaxation via increased ROS generation[19]. Several studies have documented that smoking increases the risk of developing urinary tract cancers[20-22] and the risk for end-stage renal failure in patients with renal disease[23]. Recently, several studies have shown that resveratrol exerts protective effects on acute nephrotoxicity in rats[24-27] and mice[28] by suppressing the inflammatory processes and by inhibiting lipid peroxidation. Moreover, resveratrol has a positive effect on male reproductive function by triggering penile erection, as well as enhancing blood testosterone levels, testicular sperm counts, and epididymal sperm motility[29].
In the light of these findings, we designed this study to investigate the possible protective effects of resveratrol treatment on nicotine-induced oxidative damage in rat urinary system by determining biochemical and histopathological parameters of oxidant tissue and by carrying out functional experiments to evaluate smooth muscle contractility.
Methods
AnimalsMale Wistar albino rats (300-350 g) obtained from Marmara University, Animal House were kept in a light- and temperature- controlled room with 12:12-h light–dark cycles, where the temperature (22 ± 0.5 ºC) and relative humidity (65–70 %) were kept constant. The animals were fed a standard pellet. The study was approved by the Marmara University Animal Care and Use Committee.
Experimental groups
Since nicotine is a major component of cigarette smoke, rats
were injected with nicotine to mimic the effects of chronic exposure
to nicotine. Rats in the control and nicotine-treated
groups were injected intraperitoneally (ip) with either saline
or nicotine hydrogen bitartarate (0.6 mg/kg/day; Sigma
Chemical Co., St. Louis, Missouri, USA), respectively. Along
with saline or nicotine injections that were continued for 28
days, either resveratrol (99% purity; Mikrogen Pharmaceuticals,
Turkey; RVT; 10 mg/kg/day) or saline was administered
daily by intragastric gavage. The dose and duration of nicotine
and resveratrol treatments are based on our previous studies[6-8,28]. Each experimental subgroup consisted of 16 rats.
On the 29th day of the treatments, rats were decapitated; trunk blood was collected and kidney, urinary bladder and corpus cavernosum tissues were excised. In half of all the experimental groups, corpus cavernosum and bladder were immediately prepared for the in vitro contractility studies. In the other half of the groups, corpus cavernosum, bladder and kidney samples were stored at −80 ºC until the measurement of malondialdehyde (MDA), glutathione (GSH), and luminol-lucigenin chemiluminescence (CL) levels. Extra tissue samples were fixed in 10 % buffered formalin solution and prepared for routine paraffin embedding for histological analysis.
Blood Assays
In blood samples, levels of blood urea nitrogen (BUN), creatinine,
8-hydroxy-2′-deoxyguanosine (8-OHdG), pro-inflammatory
cytokines, as well as lactate dehydrogenase (LDH) activity
and plasma total antioxidant capacity (AOC) were analyzed.
Blood urea nitrogen[30], plasma creatinine[31] concentrations
and LDH levels[32] were determined spectrophotometrically
using an automated analyzer (Bayer Opera biochemical analyzer).
Plasma levels of pro-inflammatory cytokines, tumor
necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β were quantified using enzyme-linked immunosorbent assay
(ELISA) kits specific for the previously mentioned rat cytokines
according to the manufacturer’s instructions and guidelines
(Biosource Europe S.A., Nivelles, Belgium). The total antioxidant
capacity (AOC) in plasma was measured by using a
colorimetric test system (ImAnOx, cataloge no.KC5200, Immunodiagnostic
AG, D-64625 Bensheim) according to the instructions
provided by the manufacturer. As an indicator of oxidative
DNA damage, 8-hydroxy-2′-deoxyguanosine (8-OHdG)
content in the extracted DNA solution were determined by
ELISA method (Highly Sensitive 8-OHdG ELISA kit, Japan Institute
for the Control of Aging, Shizuoka, Japan). These particular
assay kits were selected because of their high degree of
sensitivity, specificity, inter- and intraassay precision and
small amount of plasma sample required conducting the assay.
In vitro organ bath experiments
In order to assess the function of the bladder and corpus cavernosum,
contractile responses of these tissues were studied in
in vitro conditions .The bladder dome was immediately removed
and separated from the bladder base at the level of urethral
orifices and longitudinal strips of the posterior of the
bladder dome (1.5 x 5 mm) were prepared. Corpus cavernosum
excised from the penis of the rats was dissected free of the
tunica albuginea and cut into 2 x 2 x 15 mm strips. Corporeal
strips were bathed in Krebs-bicarbonate buffer containing 118
mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 25 mM NaHCO3, 1.2
mM MgSO4, 1.2 mM KHPO4 and 11.1 mM glucose, whereas
bladder strips were bathed in Tyrode’s solution containing
124.9 mM NaCl, 2.6 mM KCl, 23.8 mM NaHCO3, 0.5 mM
MgCl2, 0.4 mM NaH2PO4, 1.8 mM CaCl2 and 5.5 mM glucose.
The strips were mounted in 20 ml double-jacketed organ baths
that were aerated with 95 % O2 and 5 % CO2 (pH 7.4) at 37 ºC.
The tissues were equilibrated for 40 min under a resting tension
of 1 g. Isometric contractions were recorded using a Model
FT03 force displacement transducer (Grass Instruments,
Quincy, Massachusetts, USA) coupled to a Model 7 polygraph
(Grass Instruments). After the equilibration period, the tissues
were first exposed to KCl for maximal contraction, then the
contraction or relaxation responses were studied accordingly.
The contractile responses of the corporeal strips to 10–8 to 10–4 M phenylephrine were obtained cumulatively and expressed as the percentage of the maximal contraction induced by 124 mM KCl. After a 30-min washout period, corporeal tissues were contracted with a submaximal dose (10–5 M) of phenylephrine. The relaxation responses of the pre-contracted tissues were evaluated by adding increasing concentrations of CCh (10–8 to 10-4 M). Following another washout period, sodium nitroprusside was added cumulatively (10–-8 –10–3 M) to the corpus cavernosum strips that were pre-contracted with the same submaximal dose of phenylephrine.
The contractile responses of the bladder strips to 10-8 to 10-4 M carbachol (CCh) were obtained cumulatively and expressed as the percentage of the maximal contraction induced by 80 mM KCl. After a 30-min washout period, the bladder strips, which were precontracted with the submaximal dose of CCh (3x10-6 M), were relaxed by isoproterenol (10-10 to 10-4 M) and the relaxation responses were expressed as the percent of the contraction caused by submaximal CCh.
MDA and GSH assays
Tissue samples were homogenized with ice-cold 150 mM KCl
for the determination of MDA and GSH levels. The MDA levels
were assayed for products of lipid peroxidation[33] and
results are expressed as nmol MDA/g tissue. GSH was determined
by the spectrophotometric method, based on the use of
Ellman’s reagent[34] and results are expressed as μmol GSH/
g tissue.
MPO activity
MPO activity in tissues was measured in a procedure similar
to that documented by Hillegas et al.,[35]. Tissue samples
were homogenized in 50 mM potassium phosphate buffer (PB,
pH 6.0), and centrifuged at 41,400 g (10 min); pellets were suspended
in 50 mM PB containing 0.5 % hexadecyltrimethylammonium
bromide (HETAB). After three freeze and thaw cycles,
with sonication between cycles, the samples were centrifuged
at 41,400 g for 10 min. aliquots (0.3 ml) were added to 2.3
ml of reaction mixture containing 50 mM PB, o-dianisidine,
and 20 mM Hsub>2O2 solution. One unit of enzyme activity was
defined as the amount of the MPO present that caused a change
in absorbance measured at 460 nm for 3 min. MPO activity was
expressed as U/g tissue.
Chemiluminescence (CL) assay
To assess the role of reactive oxygen species in nicotine-induced
tissue damage, luminol and lucigenin chemiluminescences were
measured as indicators of radical formation. Lucigenin (bis-Nmethylacridiniumnitrate)
and luminol (5- amino-2,3-dihydro-
1,4-phthalazinedione) were obtained from Sigma (St Louis, MO,
USA). Luminol detects a group of reactive species, i.e.•OH,
H2O2, HOCl radicals, while lucigenin is selective for O¯2. Measurements
were made at room temperature using Junior LB 9509
luminometer (EG&G Berthold, Germany). Specimens were put
into vials containing PBS-HEPES buffer (0.5 M PBS containing
20 mM HEPES, pH 7.2). Reactive oxygen species were quantitated
after the addition of either lucigenin or luminol as an enhancer
for a final concentration of 0.2 mM. Counts were obtained
at 1 min intervals and the results were given as the area
under curve (AUC) for a counting period of 5 min. Counts was
corrected for wet tissue weight (rlu/mg tissue)[36].
Histological analysis
Kidney, bladder and corpus cavernosum samples were prepared
for routine light microscopic examination. Paraffin sections
were stained with Hematoxylin & Eosin (H&E) and examined
under an Olympus BH-2 (Tokyo, Japan) photomicroscope
by an experienced histologist who was unaware of the
treatment conditions.
Statistics
Statistical analysis was carried out using GraphPad Prism 3.0
(GraphPad Software, San Diego; CA; USA). All data are expressed
as means ± SEM. Groups of data were compared with
an analysis of variance (ANOVA) followed by Tukey’s multiple
comparison tests. Values of p<0.05 were regarded as significant.
Results
Effect of resveratrol on nicotine-induced alterations in renal function and in plasma oxidant-antioxidant statusChronic exposure to nicotine elevated plasma BUN and creatinine levels significantly (p<0.001) in the orally saline-treated group as compared to control groups (Table 1). In the group where RVT was given orally along with nicotine injections, changes in BUN and creatinine were abolished (p<0.01) and the values were not different than those of the control animals.
As an indicator of generalized tissue damage due to chronic nicotine administration, plasma LDH activity showed a significant increase (p<0.001) and this effect was reversed significantly by RVT treatment (p<0.001; Table 1). Similarly, in the plasma of saline-treated nicotine group, oxidative DNA marker 8-OHdG was increased (p<0.001), while AOC was significantly (p<0.001) depressed. On the other hand, RVT treatment reduced the plasma 8-OHdG level significantly (p<0.001) and prevented the reduction in AOC (p<0.001). Additionally, in the saline-treated group with chronic nicotine exposure, the plasma levels of pro-inflammatory cytokines, TNF-α, and IL- 1β, were significantly increased (p<0.001) when compared to control groups, while nicotine-induced elevations in the cytokine levels were significantly abolished when nicotine treatment was accompanied by RVT administration (p<0.001).
Effect of resveratrol on nicotine-induced alterations in
corpus cavernosum contractility
In the corpus cavernosum strips of the control group, which
were pre-contracted with 124 mM KCl, cumulatively added
phenylephrine (10–8 to 10–4 M) caused a concentration-dependent
contraction with an EC50 of 3.86 x 10-6 M (Fig. 1a). In the
chronically nicotine-injected group with oral saline treatment,
the contractile response of corpus cavernosum to phenylephrine
was decreased without causing any significant effect on EC50
(EC50= 3.90 x 10-6 M). However, in RVT- treated nicotine group,
the contractile response of the corpus cavernosum was higher
than in the saline-treated nicotine group, resulting in an EC50 of
1.42 x 10-6 M. The contractile responses of the RVT- and salinetreated
control groups were not different from each other.
CCh added cumulatively (10–8 –10–4 M) to corporeal tissues, which were pre-contracted with the submaximal dose of phenylephrine (10–5 M), caused a dose-dependent relaxation response in the control group (ED50= 1.2 x 10-5 M) (Fig. 1b). In the saline-treated nicotine group, relaxation response of corpus cavernosum strips to CCh was markedly reduced (ED50= 4.43 x 10-6 M) as compared to control groups. In the RVT-treated nicotine group, however, the relaxation response was higher (ED50= 6.97 x 10-6 M) than that of the saline-treated nicotine group.
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FIGURE 1: Concentration- response curve obtained by cumulative addition of a) phenylephrine b) carbachol (CCh) and c) sodium nitroprusside to corpus cavernosum strips. Values are shown as mean ± SEM of eight experiments. *p<0.05, **p<0.01, ***p<0.001 compared with saline-treated control group. +p<0.05, compared with saline-treated nicotine group. Data are given as mean ± SEM and each group consists of 8 rats. |
Sodium nitroprusside, when added cumulatively (10–-8 –10–3 M) to strips that were pre-contracted with the submaximal dose of phenylephrine, dose-dependent relaxation responses were obtained. Although this direct smooth muscle relaxant caused a similar relaxation response in the corporeal tissues of all groups (Fig. 1c), the relaxation response of the saline-treated nicotine group was significantly lower at certain concentrations (ED50= 1.39 x 10-5 M) from that of the saline-treated control group (ED50= 7.23 x 10-6 M), and this effect was reversed in the nicotine group that was treated with RVT (ED50 =1.05 x 10-5 M).
Effect of resveratrol on nicotine-induced alterations in
bladder contractility
Similar to corpus cavernosum results, cumulatively added
CCh in the saline-treated control group caused a concentration-
dependent contraction in bladder strips that were precontracted
with KCl (EC50= 1.03 x 10-6 M) (Fig. 2a). In the saline-
treated nicotine group, the contraction response of the
bladder strip to CCh was decreased (EC50= 8.39x 10-7 M), while in the RVT-treated nicotine group, the reduction in the contractile
response was relatively less (EC50 = 7.80 x 10 -7-6 M CCh) bladder strips were relaxed
in a concentration-dependent manner by the addition of
isoproterenol (10–-10 –10–4 M). The ED50 value of saline-treated
nicotine group (ED50 = 2.01 x 10-8 M) was significantly lower
than that of the control group (ED50 = 8.1 x 10-8 M), while this
decreased relaxation response was reversed in the RVT-treated
nicotine group (ED50 = 6.61 x 10-8 M). (Fig. 2b)
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FIGURE 2: Concentration- response curve obtained by cumulative addition of a) carbachol and b) isoproterenol to bladder strips. Values are shown as mean ± SEM of eight experiments. *p<0.05, ***p<0.001 compared with saline-treated control group. +p<0.05, ++p<0.01 compared with saline-treated nicotine group. Data are given as mean ±SEM and each group consists of 8 rats. |
Effect of resveratrol on nicotine-induced oxidative tissue
damage
The levels of TBARS, which is a major degradation product of
lipid peroxidation, were significantly increased in kidney,
bladder and corpus cavernosum tissues of saline-treated nicotine
group, compared with the control groups (p<0.001), while
RVT treatment in the other nicotine group caused marked decreases
in the MDA levels of all tissues (p<0.01, Fig. 3). Accordingly,
chronic nicotine administration caused significant decreases
in the tissue GSH levels of kidney, bladder and corpus
cavernosum (p<0.01). However, in the RVT-treated nicotine
group, the depletion of GSH in all tissues was prevented
(p<0.05-0.01, Fig. 4). Also, MPO activity-an index for neutrophil
infiltration was increased in chronic nicotine treatment
groups. The increase was reversed by resveratrol supplementation
(Fig. 5).
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FIGURE 3: Malondialdehyde (MDA) levels in the kidney (a); bladder (b); and corpus cavernosum (c) tissues of saline or resveratrol (RVT) treated control and nicotine groups. *** p<0.001; compared to control group; ++p<0.01 compared to saline-treated nicotine group. Data are given as mean ± SD and each group consists of 8 rats. |
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FIGURE 4: Glutathione (GSH) levels in the kidney (a); bladder (b); and corpus cavernosum (c) tissues of saline or resveratrol (RVT) treated control and nicotine groups. ** p<0.01; compared to control group; +p<0.05, ++p<0.01 compared to saline-treated nicotine group. Data are given as mean ± SD and each group consists of 8 rats. |
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FIGURE 5: Myeloperoxidase (MPO) activity in the kidney (a); bladder (b); and corpus cavernosum (c) tissues of saline or resveratrol (RVT) treated control and nicotine groups. *p<0.05, +++p<0.001; compared to control group; ++p<0.01, +++p<0.001 compared to saline-treated nicotine group. Data are given as mean ± SD and each group consists of 8 rats. |
Effect of resveratrol on nicotine-induced generation of
reactive oxygen species
Luminol and lucigenin chemiluminescences in kidney, bladder
and corpus cavernosum tissues of the saline-treated nicotine
group were significantly (p<0.05 and p<0.001) higher than
those of the controls indicating enhanced generation of reactive
oxygen species in these tissues. On the other hand, luminol
and lucigenin chemiluminescence in these tissues were
significantly (p<0.05-0.01) suppressed in the other nicotine
group that was treated with RVT (Fig. 6).
Click Here to Zoom |
FIGURE 6: Luminol and lucigenin chemiluminescence (CL) levels in the kidney (a and b); bladder (c and d); and corpus cavernosum (e and f) tissues of saline or resveratrol (RVT) treated control and nicotine groups. *p<0.05, *** p<0.001; compared to control group; +p<0.05, ++p<0.01 compared to saline-treated nicotine group. Data are given as mean ± SD and each group consists of 8 rats. |
Histological findings
The light microscopic evaluation of the kidneys in the salinetreated
nicotine group showed extensive degeneration with
severe vasocongestion in the parenchyma, wide dilatations
around the glomeruli, and vacuolizations and debris in the tubules,
while a regular morphology of the renal parenchyma
with well-designated glomeruli and tubuli were observed in
both control groups (Fig. 7a). In the kidneys of the animals
treated with nicotine and resveratrol, vasocongestion in the
parenchyma was no longer present and the glomeruli maintained
a better morphology with a mild degeneration confined
to the proximal tubuli (Fig. 7b and 7c.
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FIGURE 7: Photomicrographs of kidney revealed regular structue of glomeruli (arrow) and tubuli (*) in the saline-treated control group (a), severe vascular congestion in the interstitium (arrow) and degenerated glomeruli and tubuli (double-headed arrows) in the saline-treated nicotine group (b), and a better morphology in the glomeruli (arrow) and tubuli (double-headed arrows ) with mild interstitial congestion in the resveratrol-treated nicotine group (c). HE stain, Original magnification X 200. |
Histopathological evaluation of the urinary bladders in the control group revealed a regular mucosal layer with an overlying mucus coat, whereas an extensive surface urothelial cell detachment with a nude lamina propria and interstitial edema of the connective tissue layer was observed in the bladders of the saline-treated nicotine group (Fig. 8a, 8b). In the RVT-treated nicotine group, the urothelium appeared to gain its integrity but a mild interstitial edema was still present (Fig. 8c).
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FIGURE 8: Photomicrographs of bladder revealed regular structure of the urothelial mucosa (arrows) in the saline-treated control group (a), extensive detachment of surface urothelial cells (arrows) vasocongestion in the lamina propria in the saline-treated nicotine group (b), and reversal of degenerated epithelium with regular contours (arrows) in the resveratrol-treated nicotine group, note the persistent vasocongestion and interstitial edema (c). HE stain, Original magnification X 200 |
As compared to the regular morphology of corpus cavernosum present in the control group (Fig. 9a), chronic nicotine exposure accompanied with saline treatment led to moderate degenerations specifically in the capillaries of corpus cavernosum (Fig. 9b). Constriction of capillaries with cork-screw shaped nuclei was observed in many areas, while congestion of blood vessels was another feature noticed in the tissues. In the nicotine group that was also treated with resveratrol, the congestion of the blood vessels was reduced and the nuclei of the endothelium showed a nearly regular appearance (Fig. 9c).
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FIGURE 9: Photomicrographs of corpus cavernosum revealed a regular appearance of cavernous tissue with non-congestive vascular areas (arrows), nucleus of endothelium (inset, arrowhead) in the saline-treated control group (a); moderate degree of congestion in vascular areas (arrows) with cork-screw shaped nucleus (inset, arrowhead) in the saline-treated nicotine group (b); reduced congestion of vascular areas (arrow) and nearly-regular shape of nuclei (inset, arrowhead) in the resveratroltreated nicotine group (c). HE stain, original magnification X200, insets X400. |
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