Summary
Bu çalışmada naftalen aracılı toksisitede serbest radikallerin rolü ve erdosteinin koruyucu etkisinin incelenmesi amaçlanmıştır. Dişi Sprague-Dawley sıçanlara mısır yağ içerisinde hazırlanan 1100 mg/kg naftalen tek doz oral olarak uygulandı. Naftalen uygulamasından once 3 gün boyunca 50 mg/kg dozunda oral olarak erdostein verildi ve sıçanlar naftalen uygulamasından 24 saat sonra dekapite edildi. Karaciğer ve böbrek dokularında malondialdehit (MDA), glutatyon (GSH), düzeyleri Na+, K+-ATPaz ve myeloperoksidaz (MPO) aktiviteleri incelendi. Serum örneklerinde aspartat aminotransferaz (AST), alanin aminotransferaz (ALT), kan üre azotu (BUN), kreatinin düzeyleri ve laktat dehidrogenaz (LDH) aktivitesi ölçüldü. Plazma örneklerinde TNF-α, IL-1β, IL-6, 8-hidroksi-2'-deoksiguanozin (8- OHdG) ve total antioksidan kapasitesi (AOC) değerlendirildi. Naftalen uygulaması doku GSH düzeylerinde, Na+, K+-ATPaz aktivitesinde ve plazma AOC düzeylerinde anlamlı olarak azalmaya, doku MDA düzeylerinde ve MPO aktivitesinde anlamlı olarak artışa neden oldu. Ayrıca pro-inflamatuvar mediyatörler (TNF-α, IL-β, IL-6) 8-OHdG, LDH aktivitesi, AST, ALT, kreatinin ve BUN düzeyleri naftalen grubunda anlamlı olarak yükseldi. Buna karşılık erdostein uygulaması naftalenin neden olduğu tüm biyokimyasal değişiklikleri anlamlı olarak önledi. Sonuç olarak erdosteinin nötrofil infiltrasyonunu inhibe ederek, oksidan-antioksidan dengeyi sağlayarak ve inflamatuvar mediyatörlerin salıverilmesini düzenleyerek doku koruyucu etki gösterdiği düşünülmektedir.Introduction
Polycyclic aromatic hydrocarbons (PAHs) are the main toxic and persistent compounds present in most crude oils. As a result of the release of petroleum oils to the sea PAHs are now ubiquitous contaminants of aquatic ecosystems[1]. Some of these chemicals have been demonstrated to have mutagenic/carcinogenic[2,3], genotoxic[4] and cytotoxic[5] properties[6]. Naphthalene, a congeneric form of polycyclic aromatic hydrocarbons (PAHs), which is widely used commercially in moth repellents, lavatory scent disc and soil fumigants. It is also used in the manufacturing of naphthylamines, anthranilic and phthalic acids, and synthetic resins[7,8].The toxic manifestation induced by naphthalene appears to involve the conversion of naphthalene to naphthoquinone, as well as hydroxylated products including 1-naphthol, 2- naphthol and 1,2-dihydroxynaphthalene[9,10] which cause oxidative damage. It has been demonstrated that naphthalene exposure resulted in elevated levels of serum and liver lipid peroxides[11], and decreased hepatic selenium dependent glutathione peroxidase activity[12]. Naphthalene exposure is associated with the development of hemolytic anemia in humans and rats. Similarly administration of naphthalene (1100 mg/kg) to female Sprague-Dawley rats resulted in 2.5-fold increases in lipid peroxidation in liver and brain mitochondria 24 h after treatment indicated that the toxicity of naphthalene is at least in part related to free radicals and free radical-mediated oxidative stres[8].
Erdosteine [N-(carboxymethylthioacetyl)-homocysteine thiolactone] is a novel mucoactive agent that contains two blocked sulphydryl groups, one of which is present in an aliphatic side-chain and the other is enclosed in the heterocyclic ring[13]. These chemically blocked sulfhydryl groups are liberated following hepatic metabolism and thereby the molecule subsequently exerts its free radical scavenging and antioxidant properties[14]. Based on its free radical scavenging activity, its protective effects against oxidant-induced tissue damage have been demonstrated in various inflammation models[15-18]. Similarly we have also demonstrated the protective effect of erdosteine against colitis induced oxidative colonic tissue injury[19]. On the basis of this background, using biochemical examination, we aimed to study the putative protective effects of erdosteine on the hepatic and renal tissues in the rats exposed to naphthelene.
Methods
AnimalsAll experimental protocols were approved by the Marmara University School of Medicine Animal Care and Use Committee. Female Sprague-Dawlwey rats (200-250 g) were kept at a constant temperature (22 + 1º C) with 12 h light and dark cycles and fed a standard rat chow.
Experimental Design
Rats were given orogastrically either erdosteine (50mg/kg/ml
n=16) or saline (n=16) for 3 consecutive days. On the fourth
day, after an overnight fasting with free access to water, half of
the saline or erdosteine-treated rats were given 1100mg/kg/
ml of naphthalene in corn oil by gavage(naphtehelen groups),
while the other half of the saline or erdosteine-treated group
was given orogastrically corn oil (control groups). All rats
were decapitated at 24 hour of naphthalene or corn oil administration.
After decapitation of the animals, trunk blood was
collected and liver and kidney were carefully dissected and
stored at –70 C º for the determination of tissue malondialdehyde
(MDA) and glutathione (GSH) levels, Na+-K+ ATPase
and myeloperoxidase (MPO) activities.
Biochemical analysis
Blood urea nitrogen[20] and serum AST, ALT[21] and creatinine[22] concentrations and LDH levels[23] were determined
spectrophotometrically using an automated analyzer. Plasma
levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-
1β, and IL-6 were quantified according to the manufacturer's
instructions and guidelines using enzyme-linked immunosorbent
assay (ELISA) kits specific for the previously mentioned
rat cytokines (Biosource International, Nivelles, Belgium).
The total antioxidant capacity in plasma were measured
by using colorimetric test system (ImAnOx, cataloge no.
KC5200, Immunodiagnostic AG, D-64625 Bensheim), according
to the instructions provided by the manufacturer. The 8-
OHdG content in the extracted DNA solution were determined
by enzyme-linked immunosorbent assay (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 intra-assay precision, and small amount of
plasma sample required conducting the assay.
Malondialdehyde and glutathione assays
Tissue samples were homogenized with ice-cold 150 mM KCl
for the determination of malondialdehyde (MDA) and glutathione
(GSH) levels. The MDA levels were assayed for products of
lipid peroxidation by monitoring thiobarbituric acid reactive
substance formation as described previously[24]. Lipid peroxidation
was expressed in terms of MDA equivalents using an
extinction coefficient of 1.56 x 105 M–1 cm –1 and results are expressed
as nmol MDA/g tissue. GSH measurements were performed using a modification of the Ellman procedure[25]. Briefly,
after centrifugation at 3000 rev./min for 10 min, 0.5 ml of
supernatant was added to 2 ml of 0.3 mol/l Na2HPO4.2H2O solution.
A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml
1% sodium citrate) was added and the absorbance at 412 nm
was measured immediately after mixing. GSH levels were calculated
using an extinction coefficient of 1.36 x 104 M–1 cm –1.
Results are expressed in μmol GSH/g tissue.
Myeloperoxidase activity
Myeloperoxidase (MPO) is an enzyme that is found predominantly
in the azurophilic granules of polymorphonuclear leukocytes
(PMN). Tissue MPO activity is frequently utilized to
estimate tissue PMN accumulation in inflamed tissues and
correlates significantly with the number of PMN determined
histochemically in tissues. MPO activity was measured in tissues
in a procedure similar to that documented by Hillegass et
al.[26]. 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 H2O2 solution. One unit of
enzyme activity was defined as the amount of MPO present
that caused a change in absorbance measured at 460 nm for 3
min. MPO activity was expressed as U/g tissue.
Measurement of Na+,K+-ATPase activity
Measurement of Na+,K+-ATPase activity is based on the measurement
of inorganic phosphate that is formed from 3 mM
disodium adenosine triphosphate added to the medium during
the incubation period[27]. The medium was incubated in
a 37º C water bath for 5 min with a mixture of 100 mM NaCl, 5
mM KCl, 6 mM MgCl2, 0.1 mM EDTA, 30 mM Tris HCl (pH
7.4). Following the preincubation period, Na2ATP, at a final
concentration of 3 mM was added to each tube and incubated
at 37º C for 30 min. After the incubation, the tubes were placed
in an ice bath, and the reaction was stopped. Subsequently, the
level of inorganic phosphate was determined in a spectrophotometer
(Shimadzu, Japan) at excitation wavelength of 690 nm.
The specific activity of the enzyme was expressed as μμοl Pi
mg-1 protein h-1. The protein concentration of the supernatant
was measured by the Lowry method[28].
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 3.0
(GraphPad Software, San Diego; CA; USA). All data were 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
As shown in the AST and ALT levels in the saline treated naphthalene group were found to be significantly higher that of saline treated control group (p<0.001), however treatment with erdosteine caused significant reduction in both AST and ALT levels (p<0.01-0.05). Similarly, BUN and creatinine levels, which significantly increased in the saline treated naphthalene group (p<0.001-0.01), were also reversed back to the control levels by erdosteine treatment (p<0.01-0.05). Serum lactate dehydrogenase activity, as a marker of generalized tissue damage, showed a significant increase in the saline treated naphthalene group (p<0.001), while erdosteine administration prevented this effect (p <0.001). In the saline-treated naphthalene group, proinflammatory cytokines, TNF-α, IL-1β and IL-6, were significantly increased (p<0.001) when compared to control group, while this naphthalene-induced rise in serum cytokine levels were abolished (p<0.001) with erdosteine treatment. Plasma 8-OHdG levels, as a marker of oxidative DNA damage, were significantly higher in the saline treated naphthalene group than in control groups (p<0.001) and erdosteine treatment reduced the elevated plasma 8-OHdG levels (p<0.001). The total plasma antioxidant capacity is decreased significantly due to naphthalene administration (p<0.01), and this decrease was prevented by erdosteine treatment (p<0.05, Table 1).In accordance with these findings, the major cellular antioxidant GSH levels of liver and kidney samples in saline treated naphthalene group were significantly lower than those of the control groups (p<0.001). On the other hand, erdosteine treatment to naphthalene group restored the GSH levels in both tissues (p<0.01, Fig. 1).
Click Here to Zoom |
FIGURE 1: Glutathione (GSH) levels in the a)liver and b)kidney tissues of saline or erdosteine treated naphthalene groups and control groups. Each group consists of 8 animals. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. *** p<0.001; compared to control group. ++ p<0.01; compared to saline treated naphthalene group. |
The mean level of MDA, which is a major degradation product of lipid peroxidation, was increased in the liver and kidney tissues after naphthalene administration when compared with the control groups (p<0.001), while erdostein treatment to the naphthalene group caused a marked decrease in MDA levels (p<0.01, Fig. 2).
Click Here to Zoom |
FIGURE 2: Malondialdehyde (MDA) levels in the a) liver and b) kidney tissues of saline or erdosteine treated naphthalene groups and control groups. Each group consists of 8 animals. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. *** p<0.001; compared to control group. ++ p<0.01; compared to saline treated naphthalene group. |
Myeloperoxidase activity, as an indicator of neutrophil infiltration, was significantly higher in the liver and kidney tissues of the saline treated naphthalen group when compared to control groups (p<0.001). On the other hand, erdosteine treatment in naphthalene group significantly decreased the MPO levels both in liver and kidney (p<0.001-0.01, Fig. 3).
Click Here to Zoom |
FIGURE 3: Myeloperoxidase (MPO) activity in the liver and kidney tissues of saline or erdosteine treated naphthalene groups and control groups. Each group consists of 8 animals. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. *** p<0.001; compared to control group. ++ p<0.01, +++ p<0.001; compared to saline treated naphthalene group. |
The activity of Na+-K+-ATPase was shown to be significantly decreased in the liver and kidney tissue of saline treated naphthalen group compared with control group; however, erdosteine treatment in naphthalene group significantly increased all tissues Na+-K+-ATPase activity (p<0.001-0.01, Fig. 4).
Click Here to Zoom |
FIGURE 4: Na+-K+ ATPase activity in the a)liver and b)kidney tissues of saline or erdosteine treated naphthalene groups and control groups. Each group consists of 8 animals. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. ** p<0.01, *** p<0.001; compared to control group. ++ p<0.01, compared to saline treated naphthalene group. |
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