EXPLORING THE POTENTIAL OF ERDOSTEINE AS AN ANTIOXIDANT AGENT IN NAPHTHALENE-INDUCED OXIDATIVE STRESS: A RAT STUDY
Hariprasad Kadiyam
Asso. Professor, Department of Pharmaceutical Chemistry, Princeton College of Pharmacy, Hyderabad, Telangana, India
G Lavanya
Asst. Professor, Department of Pharmaceutical Chemistry, Princeton College of Pharmacy, Hyderabad, Telangana, India
Abstract - Erdosteine's protection from naphthalene-induced toxicity and the role of free radicals in this study are investigated. One single oral dose of corn oil containing 1100 mg naphthalene/kg was administered to female Sprague-Dawley rats. Before administering naphthalene, rats received 50 mg/kg/day of Erdosteine orally for three days. Twenty-four hours later, naphthalene was administered to the rats. Malondialdehyde (MDA), glutathione (GSH), Na+, K+-ATPase, and myeloperoxidase (MPO) activities were measured in liver and kidney tissue samples. The serum samples were tested for aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) activity, blood urea nitrogen (BUN), creatinine, and TNF-, IL-1, IL-6, 8-hydroxy-2'-deoxyguanosine (OHdG), and total antioxidant capacity (AOC). Naphthalene administration led to significant increases in tissue MDA levels and MPO activity, as well as significant decreases in tissue GSH levels, Na+, K+-ATPase activity, and plasma AOC levels. In addition, the naphthalene group had significantly higher levels of pro-inflammatory mediators (TNF-, IL-6, and IL-6), 8-OHdG, LDH activity, AST, ALT, creatinine, and BUN. However, treatment with erdosteine stopped all of these naphthalene-induced biochemical changes. In conclusion, it appears likely that erdostein protects tissues by regulating the production of inflammatory mediators, balancing the oxidant–antioxidant status, and inhibiting neutrophil infiltration.
Keywords: Naphthalene; erdosteine; peroxidation of lipids; glutathione; myeloperoxidase.
1 INTRODUCTION
The majority of crude oils contain polycyclic aromatic hydrocarbons (PAHs), which are the most harmful and long- lasting compounds. PAHs are now widespread contaminants of aquatic ecosystems due to the release of petroleum oils into the sea. It has been demonstrated that some of these chemicals are mutagenic or carcinogenic, genotoxic, and cytotoxic. Naphthalene is a common name for the polycyclic aromatic hydrocarbons (PAHs) that are used in insect repellents, scent discs for bathrooms, and soil fumigants.
Naphthylamines, anthranilic and phthalic acids, and synthetic resins are all made with it. Naphthalene's toxic effects appear to be caused by the oxidative damage caused by hydroxylated products like 1-, 2-, and 1, 2-
dihydroxynaphthalene, as well as the conversion of naphthalene to naphthoquinone. Naphthalene exposure has been shown to decrease hepatic selenium dependent glutathione peroxidase activity and raise serum and liver lipid peroxide levels. In both humans and rats, exposure to naphthalene is linked to the development of hemolytic anemia. The fact that female Sprague- Dawley rats received 1100 mg/kg of naphthalene and experienced 2.5-fold increases in lipid peroxidation in their mitochondria in the liver and brain 24 hours after treatment suggested that the toxicity of naphthalene is at least partially related to free radicals and free radical- mediated oxidative stress.
Erdosteine (N-
(carboxymethylthioacetyl)-homocysteine
thiolactone) is a novel mucoactive agent with two blocked sulphydryl groups. One of the blocked sulphydryl groups is in an aliphatic side chain, and the other is in the heterocyclic ring. Following hepatic metabolism, these chemically blocked sulfhydryl groups are released, allowing the molecule to exercise its antioxidant and free radical scavenging capabilities.
In various models of inflammation, its protective effects against oxidant-induced tissue damage have been demonstrated based on its free radical scavenging activity. In a similar vein, we have demonstrated that erdosteine protects against oxidative colonic tissue damage caused by colitis. Based on this background, we wanted to investigate the erdosteine's alleged protective effects on the hepatic and renal tissues of naphthelene-exposed rats through biochemical analysis.
2 MATERIALS AND METHODS 2.1 Animals
All trial conventions were supported by the Marmara College Institute of Medication Creature Care and Use Board of trustees. Standard rat food was fed to 200-250 g female Sprague-Dawlwey rats during 12 h of light and dark cycles at a constant temperature of 22 + 1o C.
2.2 Experimental Design
For three days, rats were given either saline (n=16) or erdosteine (50 mg/kg/ml) orally. On the fourth day, half of the saline- or erdosteine-treated rats were given 1100mg/kg/ml of naphthalene in corn oil by gavage (naphthelen groups) following an overnight fast in which they had free access to water. The other half of the saline- or erdosteine-treated rats were given corn oil orally (control groups). After being given naphthalene or corn oil for 24 hours, all of the rats had their heads cut off. Tissue malondialdehyde (MDA) and glutathione (GSH) levels, as well as the activities of Na+-K+ ATPase and myeloperoxidase (MPO), were measured
by carefully dissecting and storing the liver and kidney at –70°C following the decapitation of the animals.
2.3 Biochemical Analysis
Using an automated analyzer, the concentrations of urea nitrogen, AST, ALT, creatinine and LDH in serum were measured spectrophotometrically. Using enzyme-linked immunosorbent assay (ELISA) kits designed specifically for the rat cytokines (Biosource International, Nivelles, Belgium), plasma levels of tumor necrosis factor alpha (TNF-), interleukin (IL)-1, and IL-6 were measured in accordance with the manufacturer's instructions and guidelines. The colorimetric test system (ImAnOx, catalog number) was used to measure the total antioxidant capacity of the plasma.
KC5200, Immunodiagnostic AG, D-64625 Bensheim), following the manufacturer's instructions. The enzyme-linked immunosorbent assay (ELISA) method (Highly Sensitive 8-OHdG ELISA kit, Japan Institute for the Control of Aging, Shizuoka, Japan) was used to measure the amount of 8-OHdG present in the extracted DNA solution. The selection of these particular assay kits was based on their high level of sensitivity, specificity, precision both inter- and intra-assay, and the small amount of plasma sample required to carry out the assay.
2.4 Malondialdehyde and Glutathione Assays
For the purpose of determining the concentrations of malondialdehyde (MDA) and glutathione (GSH), tissue samples were homogenized with ice-cold 150 mM KCl. By keeping an eye on the formation of thiobarbituric acid reactive substances, the MDA levels were checked for lipid peroxidation products. Using an extinction coefficient of 1.56 x 105 M–1 cm–1, the results of lipid peroxidation were expressed in terms of MDA equivalents. The Ellman method was modified for the purpose of taking GSH
measurements. Briefly, 2 ml of 0.3 mol/l Na2HPO4.2H2O solution was mixed with 0.5 ml of supernatant after 10 minutes of centrifugation at 3000 rev/min. The absorbance at 412 nm was measured immediately following the addition of a 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml, 1% sodium citrate). Using an extinction coefficient of 1.36 x 104 M–1 cm–1, GSH levels were determined. The measurements are given in mol GSH/g tissue.
2.5 Myeloperoxidase Activity
An enzyme known as myeloperoxidase (MPO) is primarily found in the azurophilic granules of polymorphonu clear leukocytes (PMN). The number of PMN counted histologically in tissues is strongly correlated with tissue MPO activity, which is frequently used to estimate PMN accumulation in inflamed tissues. Similar to what was described by Hillegass et al., MPO activity was measured in tissues. After being homogenized in PB (50 mM potassium phosphate, pH 6.0), tissue samples were centrifuged at 41.400 g for ten minutes;
In 50 mM PB containing 0.5%
hexadecyltrimethylammonium bromide (HETAB), pellets were suspended. The samples were centrifuged for ten minutes at 41.400 g after three freeze-thaw cycles with sonication in between. Aliquots (0.3 ml) were added to 2.3 ml of response blend containing 50 mM PB, o- dianisidine, and 20 mM H2O2 arrangement. The amount of MPO present that caused a change in absorbance measured at 460 nm for three minutes was considered to be one unit of enzyme activity. U/g tissue was used to measure MPO activity.
2.6 Measurement of Na+, K+-ATPase Activity
The inorganic phosphate that is produced when 3 mM disodium adenosine triphosphate is added to the medium during the incubation period is used as
the basis for measuring Na+,K+-ATPase activity. 100 mM NaCl, 5 mM KCl, 6 mM MgCl2, 0.1 mM EDTA, and 30 mM Tris HCl (pH 7.4) were added to the water bath to incubate the medium for five minutes.
Each tube was incubated for 30 minutes at 37 degrees Celsius with a final concentration of 3 mM of Na2ATP following the preincubation period. After the hatching, the cylinders were set in an ice shower, and the response was halted.
A spectrophotometer from Shimadzu, Japan, was used to measure the level of inorganic phosphate at an excitation wavelength of 690 nm. The enzyme's specific activity was represented as l Pi mg-1 protein h-1. The Lowry method was used to measure the supernatant's protein content.
3 DISCUSSION
The current data clearly demonstrate that treatment with erdosteine significantly reduced napthelene-induced lipid peroxidation and neutrophil infiltration in the hepatic and renal tissues due to its free radical scavenging properties, while restoring the depleted antioxidant GSH level and inhibited Na+-K+-ATPase activity to control levels. The severity of oxidative stress is also demonstrated by decreased AOC and elevated serum levels of LDH, AST, ALT, BUN creatinine, and 8-OHdG.
The outcomes likewise exhibit that erdosteine treatment, as seen by the inversion of modifications in every one of the deliberate boundaries in tissues, mitigated the naphtelene-actuated plasma markers of oxidative pressure and worked on renal and hepatic capabilities.
The neutrophil-produced free radicals may be partly to blame for this rise in lipid peroxidation. Because; In addition to their direct harm to tissues, free radicals appear to cause the accumulation of leukocytes in the affected tissue, thereby indirectly causing tissue damage through activated neutrophils.
Through the production and release of reactive oxygen metabolites and cytotoxic
proteins (like lactoferrin, proteases, and myeloperoxidase) into the extracellular fluid, activated neutrophils are known to cause tissue damage. At the point when neutrophils are invigorated by different energizers, MPO, as well as other tissue- harming substances are let out of the cells. In this manner, it is a list of neutrophil penetration. Naphthalene's increase in MPO activity may cause organ inflammation and damage because neutrophil infiltration is a crucial part of acute inflammation. In contrast, a significant increase in the proinflammatory cytokines TNF-alpha, IL- 1, and IL-6 demonstrates that naphthalene toxicity is closely linked to inflammatory mechanisms and oxidative damage. Our findings suggest that the protective effects of erdosteine are partially mediated by blocking plasma cytokines and tissue neutrophil infiltration because treatment with erdosteine significantly reduced these cytokines and prevented the infiltration of neutrophils into the damaged tissue.
Testicular torsion and detorsion in rats have also been used to investigate the effect of erdosteine. Before rats were subjected to left unilateral testicular torsion and detorsion, Erdosteine was administered at a dose of 50 mg kg-1 per day for two days. By preventing the accumulation of free oxygen radicals, erdosteine administration reduced the histological damages associated with testicular torsion and detorsion.
4 CONCLUSION
In conclusion, this study clearly demonstrates that oxidative metabolism of naphthalene is one of the primary mechanisms that causes multiple organ damage. Erdosteine's protective effects can be attributed, at least in part, to its ability to inhibit neutrophil infiltration, balance oxidant-antioxidant status, and regulate the generation of inflammatory mediators. This suggests that erdosteine may play a role in the treatment of organ
failures caused by chemical or drug toxicities.
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