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- .Original Article . -
Oxidative stress in testicular tissues of rats exposed to cigarette smoke and protective effects of caffeic acid phenethyl ester
Hüseyin Ozyurt1, Hýdýr Pekmez2, Bekir Suha Parlaktas3, Ilter Kus4, Birsen Ozyurt5, Mustafa Sarsýlmaz4
Departments of 1Biochemistry,
3Urolog and 5Anatomy, School of Medicine, Gaziosmanpasa University, Tokat 60100,
Turkey
2School of Health Sciences and
4Department of Anatomy, School of Medicine, Firat University,
Elazýð 23100, Turkey
Abstract
Aim: To show the oxidative stress after cigarette smoke exposure in rat testis and to evaluate the effects of caffeic
acid phenethyl ester (CAPE). Methods: Twenty-one rats were divided into three groups of seven. Animals in Group I
were used as control. Rats in Group II were exposed to cigarette smoke only (4×30min/d) and rats in Group III
were exposed to cigarette smoke and received daily intraperitoneal injections of CAPE
(10µmol/kg¡¤d). After 60 days all the rats were killed and the levels of nitric oxide (NO) and anti-oxidant enzymes such as superoxide-dismutase,
catalase and glutathione peroxidase (GSH-Px) and the level of malondialdehyde were studied in the testicular tissues of
rats with spectrophotometric analysis. Results:
There was a significant increase in catalase and superoxide-dismutase
activities in Group II when compared to the controls, but the levels of both decreased after CAPE administration in
Group III. GSH-Px activity was decreased in Group II but CAPE caused an elevation in GSH-Px activity in Group III.
The difference between the levels of GSH-Px in Group I and Group II was significant, but the difference between
groups II and III was not significant. Elevation of malondialdehyde after smoke exposure was significant and CAPE
caused a decrease to a level which was not statistically different to the control group. A significantly increased level
of NO after exposure to smoke was reversed by CAPE administration and the difference between NO levels in groups
I and III was statistically insignificant. Conclusion:
Exposure to cigarette smoke causes changes in the oxidative
enzyme levels in rat testis, but CAPE can reverse these harmful
effects.
Keywords: testis; cigarette; caffeic acid phenetyl ester; anti-oxidants; nitric oxide
Dr Mustafa Sarsýlmaz, M.D., Department of Anatomy, School of Medicine, Firat University, Elazýð 23100, Turkey.
Tel: +90-532-411-2110, Fax: +90-424-237-9138
E-mail: msarsilmaz@firat.edu.tr
Received 2005-08-09 Accepted 2005-12-06
1 Introduction
Cigarette smoke could cause negative effects on eve-ry system in an organism due to the various toxic substances
and pro-oxidizing and oxidizing materials found in its structure. Free radicals (FRs) continuously arise in the body as
a result of ongoing chemical events, such as oxidative phosphorylation, uric acid metabolism and prostaglandin
synthesis [1]. The cellular anti-oxidant defense system consists of superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GSH-Px) and melatonin. Vitamin E
(á-tocopherol), â-carotene, vitamin C, flavonoids, and
coenzymes Q and A are also anti-oxidant factors. The shift of the delicate balance between FRs and the cellular
anti-oxidant defense system in favor of FRs might lead to development of oxidative stress [1]. Increased activity of free
radical scavenger enzymes, such as SOD, CAT and GSH-Px, to prevent the detrimental effects of oxidative stress in
different organs has been shown in published reports [2].
Testicular tissue is a highly vascular tissue, and because of the rich blood supply, cigarette smoke may deteriorate
the balance between oxidant and anti-oxidant enzyme systems. As a result, FRs are generated, which cause testicular
tissue damage. In a recent study it was shown that oxidative stress causes the production of abnormal spermatozoa
and affects sperm functions [3].
Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis and it is known to have
powerful antimicrobial, anti-inflammatory, antineoplastic and anti-oxidizing effects [4]. CAPE exerts its bene-ficial effects
by decreasing free oxygen radicals, and prevents consumption of FR scavenging enzymesacting in parallel to these
anti-oxidant enzymes [5, 6]. According to previous studies, CAPE exerts its beneficial effects by decreasing free
oxygen radicals and prevents consumption of free radical scavenging enzymes acting in parallel to these antioxidant
enzymes [4].
The male factors in fertility assessment are usually based on the evaluation of several semen parameters [7]. It is
well known that these parameters are affected by factors such as smoking [7]. Histologic changes in the seminiferous
tubular structure, sperm counts and morphology have been mentioned in related articles [8]. But the oxidative stress
due to cigarette smoke and the effects of CAPE on this condition have not been studied.
In this experimental study, we aimed to show the oxidative stress exerted on rat testicular tissue due to cigarette
smoke and to show the effects of intraperitoneal injection of the anti-oxidizing agent CAPE against this condition.
2 Materials and methods
2.1 Animals
Twenty-one male Wistar-Albino rats (weighing 200-250g) were used in the study. There were three
experimental groups, each consisting of seven rats. The rats in the control group (Group I) breathed normal clean air. In Group
II, rats breathed cigarette smoke only
(4×30min/d). The rats in Group III received daily intraperitoneal injections of
CAPE (10µmol/kg¡¤d) with cigarette smoke exposure. International standarts for the care of laboratory animals were
followed and the protocol of the study was approved by the responsible local ethical commitee.
2.2 Cigarette smoking and isolation of organ
The second-hand subjection of each rat to cigarette smoke inhalation was achieved in a glass cabin
(100×50×20cm) with the help of an aquarium air pump. One end of a plastic tube was implanted into the air pump
and placed into the glass container with a cigarette on the other end. Non-filtered commercially available cigarettes
(Birinci-Tekel) were used. Two lit cigarettes were placed in the cages four times a day, for 30min each period, during
the study period (groups II and III). CAPE (10µmol/kg) was injected intraperitoneally once per day into each rat in
Group III. Control rats in Group I breathed normal air. At the end of the experimental period (60 days) the rats were
decapitated. Testes of all rats were removed with standart
mid-scrotal incisions and frozen immediately in a deep
freezer (-85°C).
All of the testicular tissues were washed with cold saline solution and wet tissue weights were obtained. The tissues
were cut into small pieces and placed into glass bottles. They were then homogenized in ice-cold Tris-HCl buffer solution
(0.2mmol/L and 50/39.9 [v/v]), within a homogenizer (Ultra Turrax Type T25-B; IKA Labortechnic, Staufen, Germany)
for 2min at 11 200 ×g. The homogenate was centrifuged at 3
500×g for 60min and a supernatant was obtained.
The levels of CAT and GSH-Px were determined in the supernatant, and NO and malondialdehyde (MDA) levels were
studied in the homogenate. For a further extraction procedure, the supernatant was extracted in ethanol/chloroform
mixture (5/3, v/v). After a second centrifugation at 3
500×g (20min), the clear upper layer (the ethanol phase) was
taken and used in SOD activity determination. All procedures were performed at
4°C and icepacks were used to maintain the temperature during the homogenization procedure.
2.3 SOD activity determination
The principle of the SOD activity determination method is based on the inhibition of nitroblue tetrasolium
reduction by the xanthine-xanthine oxidase system as a superoxide radical generator. One unit of SOD was defined as the
enzyme activity causing 50% inhibition in the nitroblue tetrazolium reduction rate. SOD activity was expressed as
units per mg tissue protein (U/mg prot).
2.4 CAT activity determination
The essentials of the CAT activity determination method were based on the determination of the rate constant of
the H2O2 decomposition rate at 240nm. The essentials CAT activity determination method were based on the
determination of the rate constant of the
H2O2 decomposition rate at 240 nm. Results were expressed as U/mg
prot.
2.5 GSH-Px activity determination
GSH-Px was measured by the enzymatic reaction which was initiated by addition of
H2O2 to the reaction mixture
containing reduced glutathione, NADPH and glutathione reductase and the change in the absorbance at 340nm was
monitored by spectrophotometer. Activity was given in U/mg prot.
2.6 MDA level determination
Testicular tissue MDA levels were analyzed by a method based on the reaction with thiobarbituric acid at
90-100°C. In the thiobarbituric acid test reaction, MDA or MDA-like substances and thiobarbituric acid react together to
produce a pink pigment with an absorption maximum of 532nm. The results were expressed as nanomol per gram
wet tissue (nmol/g wet tissue) calculated by using a standard graphics, which was prepared with serial dilutions of
standard 1,1,3,3-tetrame-thoxypropane.
2.7 NO determination
NO measurement is very difficult in biological spe-cimens, therefore tissue nitrite
(NO2-) and nitrate (NO3-) were
estimated as an index of NO production. Samples were initially deproteinized with Somogyi reagent. Total nitrite
(nitrite+nitrate) was measured after conversion of nitrate to nitrite by copperized cadmium granules by a
spectrophotometer at 545nm. A standard curve was established with a set of serial dilutions
(10-8-10-3mol/L) of sodium nitrite. Linear
regression was carried out using the peak area from the nitrite standard. The resulting equation was then used to calculate
the unknown sample concentrations. Results were expressed as nmol/g wet tissue.
2.8 Protein determination
Protein content in the alkaline mixture formed a complex with copper and caused reduction of
phosphomolibdate-phosphotungstate reactives. Color change in the media was proportional with the protein concentration, and
absorbance at 700nm was monitored by spectrophotometer.
2.9 Statistical analysis
Statistical analysis was carried out using SPSS version 10.0 for Windows software (SPSS, Chicago, IL, USA).
Distribution of the groups was analyzed with one sample Kolmogorov-Smirnov test. As all groups showed normal
distribution, group differences were analyzed using parametric statistical methods, paired independent sample
t-tests following one-way ANOVA. Results were presented as mean±SD.
P (0.05 was considered statistically significant.
3 Results
Oxidative stress caused by cigarette smoking and the effects of CAPE on these changes were determined by
measuring the level of SOD, CAT and GSH-Px enzyme activity, and tissue levels of NO and MDA. The results are
presented in Table 1. SOD activity in testis tissues of rats exposed to cigarette smoke only (Group II) increased
significantly compared to the control group
(P(0.05), but SOD in Group III decreased to a level closer to the
control group (P=0.05). Compared to the control group, CAT activity in Group II increased significantly
(P(0.05), but in Group III, CAT activity decreased significantly
(P(0.05). The significant decrease in GSH-Px activity in Group II
compared to Group I (P(0.05) was reversed in Group III after administration of CAPE
(P(0.05), and the difference between Group I and Group III was not significant
(P=0.05). In the evaluation of MDA, a statistically significant
increase in Group II was observed (P(0.05), but in Group III, a reduction in MDA levels was observed and the
difference with the control group was not significant
(P=0.05). The elevation of NO was significant in Group II
(P(0.05) but, with the administration of CAPE, the testicular tissue NO level in Group III was brought to a level
very close to that of the control group (P=0.05; Table 1).
4 Discussion
Smoking is a noxious process that triggers oxidative stress, not only in first-hand smokers but also in those
exposed to second-hand smoke. By-products of chemical events, namely FRs and reactive oxygen species, cause
this oxidative stress. Cigarette smoking is also known to be associated with various cardiovascular diseases and
chronic pulmonary diseases, ranging from infection to lung cancer [7-9].
FRs or reactive oxygen species have potentially harmful effects and cause oxidative stress on the metabolic
events occuring in molecules. The cellular anti-oxidant defense system controls the effects of these species and this
duty is carried by FR scavenger enzymes, such as SOD, CAT and GSH-Px [10]. When there is an impairment in the
cellular anti-oxidant defense system and/or FR production exceeds the ability of this defense system to scavenge these
species, oxidative stress occurs and FRs attack polyunsaturated fatty acids found widely in cell membranes. Lipid
peroxidation develops in cell membranes, which causes production of membrane destruction products, such as MDA
[10].
Baskaran et al. [11] studied the effects of cigarette smoke on lipid peroxidation and enzyme activities of SOD,
CAT, GSH-Px. They observed increased lipid pero-xidation in liver, lung and kidneys, and anti-oxidant enzyme
activities were also elevated in these organs. Nielsen
et al. [12] found slightly increased MDA levels in the plasma of
smokers when compared to non-smokers. They supported that the plasma MDA level may be a potential biomarker
for oxidative stress which occurred due to lipid peroxidation. In another experimental study, elevated MDA levels and
decreased GSH and GSH-Px activity were found in rat tissues after the 45-day exposure to cigarette smoke [13].
NO is a water- and lipid-soluble FR synthesized in the vascular endothelium from
L-arginine by the action of NO synthase enzymes. It plays an important role in the regulation of blood flow in normal and pathologic situations. There
could be two reasons for the elevation of tissue NO levels after exposure to cigarette smoke: increased synthesis
because of the injury of the vascular endothelium, or the activation of neutrophils in damaged testicular tissue, leading
to synthesis of NO [4]. Determination of the changes in NO levels and the effect of CAPE on NO levels after testicular
torsion and detorsion in rats was reported in an experimental study by Koltuksuz
et al. [4]. Their results showed that NO levels increased with torsion, and administration of CAPE prevented the decrease in NO levels during reperfusion.
They concluded that this effect of CAPE on NO levels could be important for protecting testis from such an
ischemia/reperfusion injury.
In previous studies, cigarette smoking has been shown to be associated with an overall decreased fertilizing
capacity of sperm [14]. The mechanisms of the decrease in male reproductive capacity due to exposure to cigarette
smoke ranges from causing erectile dysfunction by inducing some vasculogenic risk factors, to decreasing sperm
counts, affecting sperm motility and deteriorating sperm membrane characteristics [15]. Cigarette smoke also leads
to secretory dysfunction of both Sertoli and Leydig cells and causes harmful effects on the epididymal sperm
maturation process and sperm capacity to penetrate oocytes [14].
Lower seminal plasma anti-oxidant enzyme levels and increased oxidative damage to sperm DNA due to cigarette
smoke has been shown by Fraga et al. [16]. In addition, mitochondrial and nuclear DNA damage as a result of
accumulated oxidative stress and endogenous DNA strand breaks in human spermatozoa after exposure to smoke has
also been mentioned in other studies [17, 18].
It has also been shown that the serum levels of cotinine correlate with the number of cigarettes smoked, and
passage of the noxious metabolites of cigarette smoke through the blood-testis barrier has been suggested as the basis
of the oxidative stress and eventual oxidative damage of testicular tissues [19, 20].
The current study demonstrated that cigarette smoking had harmful effects on testicular tissue by generation of
free oxygen radicals. In the CAPE-administered group, the elevation in GSH-Px enzyme activity might be an
explanation for the protective effect of CAPE. The rising CAT and SOD activity and MDA levels in the rat testicular tissue due
to cigarette smoke exposure were also reversed with administration of CAPE. These results propose the anti-oxidant
effect of CAPE by direct or indirect effects on promoting the activity of anti-oxidant enzyme activities, or directly by
free oxygen radical scavenging activity. It is also apparent that this oxidative stress will have a negative impact on
male fertility potential. Further studies to show the histologic changes in sperm functions, seminiferous tubules, and
Sertoli and Leydig cell functions, which associates histopathologic effects with biochemical results, should be performed.
In conclusion, exposure to cigarette smoke results in the overproduction of reactive oxygen species in rat testis.
The administration of CAPE significantly decreases the oxidative stress on testicular tissue caused by cigarette smoke.
Thus, we can say that CAPE administration in the appropriate dosage and duration prevents the adverse oxidative
effects of cigarette smoking on testicular tissue.
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