Orhan Erdoğan

Deltamethrin attenuates antioxidant defense system and induces the expression of heat shock protein 70 in rainbow trout.

The current research aims to determine alterations in gene expression and enzymatic activity of fish antioxidant metabolism in response to pesticide administration. To this end, three different deltamethrin concentrations (0.25, 1, 2.5mug/L) were administrated to rainbow trout (Oncorhynchus mykiss) at different time intervals (6, 12, 24, 48 and 72h) in order to observe the influences of the pesticide on the activity of glutathione reductase, glucose 6-phosphate dehydrogenase, 6-ghosphogluconate dehydrogenase, and the expression of Hsp70 gene. We observed that the activities of the enzymes decreased with increasing deltamethrin concentrations and exposure time. The pesticide had more inhibitory effects on gill enzymes than those of muscle, liver and kidney. In addition, we detected that deltamethrin increased the expression of the stress-related protein Hsp70 with significant fold-chance values. The efficiency rate was 96.4% which is equal to 1.96 calculated via conversion formula used to calculate fold-chance value. We conclude that deltamethrin causes oxidative stress in fish both at protein and mRNA levels.

Purification and characterization of carbonic anhydrase from the teleost fish Dicentrarchus labrax (European seabass) liver and toxicological effects of metals on enzyme activity

Carbonic anhydrase (EC 4.2.1.1; CA) was purified and characterized from the liver of the teleost fish Dicentrarchus labrax (European seabass) for the first time. The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The enzyme was purified 78.8-fold with a yield of 46%, and a specific activity of 751.72U/mg proteins. It has an optimum pH at 7.5; an optimum temperature at 25°C; an optimum ionic strength at 10mM and a stable pH at 8.5. The kinetic parameters of this enzyme were determined for its esterase activity, with 4-nitrophenyl acetate (NPA) as substrate and the purified enzyme had an apparent K(M) and V(max) values of 0.44 mM and 0.249 μmolxmin(-1), respectively. The following metals, Al(+3), Cu(+2), Pb(+2), Co(+3), Ag(+1), Zn(+2) and Hg(+2) showed inhibitory effects on the enzyme. Al(+3) and Cu(+2) exhibited the strongest inhibitory action. Pb(+2) was moderate inhibitor, whereas other metals showed weaker actions. All tested metals inhibited the enzyme in a competitive manner. Our findings indicate that these metals inhibit the fish enzyme in a similar manner to other α-CAs from mammals investigated earlier, but the susceptibility to various metals differ between the fish and mammalian enzymes. Our results also demonstrate that these metals might be dangerous at low micromolar concentrations for fish CA enzymes.

Acute and long-term genotoxicity of deltamethrin to insulin-like growth factors and growth hormone in rainbow trout

We report here the acute and long-term influences of deltamethrin on the expression of IGF-I, IGF-II and GH-I in rainbow trout muscles. We treated rainbow trouts with different concentrations of deltamethrin (0.25 microg/L, 1 microg/L and 2.5 microg/L) and observed the alterations in mRNA expression levels of IGF-I, IGF-II and GH-I at different time intervals (at 6th, 12th, 24th, 48th, 72nd hours and 30th day). The mRNA levels significantly decreased with increasing deltamethrin concentrations for acute administration. Interestingly, a significant recovery in GH-I expression was seen after the 72nd hour up to 30th day while no significant differences were observed for IGF-I and IGF-II between the same time intervals. Here we demonstrate that deltamethrin exposure decreases the expression of IGF-I, IGF-II and GH-I in rainbow trout which might cause undesirable outcomes not only in growth, but also in development and reproduction.

Impact of deltamethrin exposure on mRNA expression levels of metallothionein A, B and cytochrome P450 1A in rainbow trout muscles

Metallothioneins (MT) are widely utilized to identify specific responses to heavy metal pollution. In addition, there is evidence demonstrating that in vertebrates MT synthesis is stimulated by different endogenous and exogenous agents in particular compounds leading to production of ROS. Also, cytochrome P450 1A can enhance the generation of ROS. On this basis, MT and CYP 1A induction can be considered as biomarkers of oxidative stress. In the current study, we examined the influences of pesticide administration on the expression of MT-A, MT-B and CYP 1A. For this purpose, we produced muscle metallothionein-A, metallothionein-B and cytochrome P450 1A cDNAs and used quantitative RT-PCR to assay mRNAs in rainbow trout exposed to acute and long-term deltamethrin administration. We observed that deltamethrin exposure significantly (p<0.05) increased the expression levels of Cyp1A, MT-A and MT-B in a time dependent manner. Results of our study contributes to the identification of inducers of such biomarkers in addition to well known agents.

Effects of glyphosate on juvenile rainbow trout (Oncorhynchus mykiss): transcriptional and enzymatic analyses of antioxidant defence system, histopathological liver damage and swimming performance.

This study aims to determine the effect of glyphosate on the transcriptional and enzymatic activity of antioxidant metabolism enzymes of juvenile rainbow trout with short term (6, 12, 24, 48 and 96 h) and long term (21 days) exposures followed by a recovery treatment. This study also aims to determine the effects of glyphosate exposure on liver tissue damage and swimming performance due to short term (2.5, 5 and 10 mg/L) and long term (2.5 and 5 mg/L) exposures. Following pesticide administration, ten fish, each as a sample, were caught at 6th, 12th, 24th, 48th and 96th -h for the short term, and at 21st day for the long term exposure study. GPx activity was found to be significantly induced 12 h after the exposure to 2.5 mg/L of glyphosate as compared with the control group. A similar degree of induction was also observed for CAT activity but not for SOD. For long term exposure, except for the GPx activity after exposure to 5 mg/L of glyphosate, the activities of all other enzymes remained on a par with the control group. It was also observed that the levels of gene expression of these enzymes were not comparable with each other. It is assumed that these differences might result from the effect of glyphosate before translation and the possible reasons for this scenario are also discussed. The results of swimming performance are found to be consistent with responses of the antioxidant system, and they are attributed to the energy metabolism. The data are also supported with liver histopathology analysis.

Expression of Glucose-6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase in Oxidative Stress Induced by Long-Term Iron Toxicity in Rat Liver

Reactive oxygen species (ROS) are highly reactive and oxygen‐containing molecules that are derived by metabolic activities or from environmental sources. Toxicity of heavy metals including iron has the ability to generate ROS in all living organisms. The pentose phosphate pathway enzymes, which are glucose 6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase, produce nicotinamide adenine dinucleotide phosphate (NADPH) that enables cells to counterbalance the oxidative stress via the action of the glutathione system. The results presented here have shown that toxic and nontoxic levels of iron have a strong effect on the expression of both genes. While toxic levels of iron exhibited significant changes in enzyme activity, nontoxic levels had no effect on enzymes in rat liver. Our results are the first evidence to elucidate how oxidative stress induced by long‐term iron toxicity affects both enzymes at the enzymatic and molecular level and also to determine any possible correlation between the enzymatic and molecular levels.

Chronic toxicity of pesticides to the mRNA expression levels of metallothioneins and cytochrome P450 1A genes in rainbow trout

The hazardous effects of pesticides on various metabolic pathways are a great problem for environmental health and should be well determined. In the present study, the authors treated rainbow trout with 0.6 μg/L deltamethrin for 28 days and 1.6 mg/L 2,2-dichlorovinyl dimethyl phosphate for 21 days. After this time period, the authors observed alterations in mRNA expression levels of MT-A, MT-B and CYP-1A. Chronic exposure to low levels of pesticides may have a more significant effect on fish populations than acute poisoning. While both pesticides caused a significant increase on mRNA levels of MT-A and CYP-1A, MT-B mRNA levels were increased significantly only upon deltamethin administration. The significant increase in mRNA levels of the corresponding genes may be considered as a defence mechanism in addition to the antioxidants against oxidative stress, as well as a detoxification mechanism against adverse effects of pesticides.

Stimulation of gene expression and activity of antioxidant related enzyme in Sprague Dawley rat kidney induced by long-term iron toxicity

The trace elements such as iron are vital for various enzyme activities and for other cellular proteins, but iron toxicity causes the production of reactive oxygen species (ROS) that causes alterations in morphology and function of the nephron. The present study was designed to determine the effect of long-term iron overload on the renal antioxidant system and to determine any possible correlation between enzymatic and molecular levels. Our data showed that reduced glutathione (GSH) levels, which is a marker for oxidative stress, strikingly decreased with a long-term iron overload in rat kidney. While renal mRNA levels of glucose 6-phosphate dehydrogenase (G6pd), 6-phosphogluconate dehydrogenase (6pgd) and glutathione peroxidase (Gpx) were significantly affected in the presence of ferric iron, no changes were seen for glutathione reductase (Gsr) and glutathione S-transferases (Gst). While the iron affected the enzymatic activity of G6PD, GSR, GST, and GPX, it had no significant effect on 6PGD activity in the rat kidney. In conclusion, we reported here that the gene expression of G6pd, 6pgd, Gsr, Gpx, and Gst did not correlate to enzyme activity, and the actual effect of long-term iron overload on renal antioxidant system is observed at protein level. Furthermore, the influence of iron on the renal antioxidant system is different from its effect on the hepatic antioxidant system.

Expression of hCA IX isoenzyme by using sumo fusion partner and examining the effects of antitumor drugs / Sumo füzyon partneri kullanarak hCA IX izoenziminin ekspresyonu ve antitümör ilaçların etkilerinin incelenmesi

Abstract Objective: In this study, investigating the effects of inhibition of the enzyme activity of some antitumor drugs and the Cancer-Related Human Carbonic Anhydrase IX (hCA IX) isoenzyme expressing as a SUMO fusion protein in an Escherichia coli expression system were aimed. Methods: hCA IX isoenzyme was expressed using SUMO fusion technology. The fusion protein was expressed in a totally soluble form and the expression was verified by SDS-PAGE analysis. Affinity chromatography was used in the purification processes. The effects of certain antitumor drugs on enzyme activity were investigated in vitro conditions by using esterase activity. IC50 values of drugs showing the inhibitory effect were calculated. Inhibition types and Ki values for antitumor drugs, which inhibit the enzyme, were determined by separately plotting Lineweaver- Burk plots. Results: The molecular weight of the fusion protein was approximately 85kDa. The optimal induction concentration of IPTG and the growth temperature were found to be 1.0mM and 30°C. The fusion protein was purified at approximately 3.07-fold with a yield of 92.58%, and a specific activity of 43707EU/mg proteins by nickel nitrilo-triacetic acid resin chromatography. Conclusion: Our work is extremely important because CA IX plays a clinical role as a biomarker in cancer diagnosis and the use of specific inhibitors of the CA IX enzyme will be useful in the fight against cancer. In vitro inhibition studies on the recombinant hCA IX enzyme can shed light on the development of anticancer drugs for cancers overexpressing CA IX. Özet Amac: Bu calışmada kanser ilişkili İnsan Karbonik Anhidraz IX (hCA IX) izoenziminin Escherichia coli ekspresyon sistemi icinde bir SUMO fuzyon proteini olarak ekspre edilmesi ve bazı antitumor ilacların enzim aktivitesi uzerine inhibisyon etkilerinin incelenmesi amaclanmıştır. Metod: hCA IX izoenzimi SUMO fuzyon teknolojisi kullanılarak ekspre edildi. Fuzyon protein tumuyle cozunur bir formda ekspre edildi ve ekspresyon SDS-PAGE analizi ile doğrulandı. Saflaştırma işlemlerinde afinite kromatografisi kullanıldı. Enzim aktivitesi uzerine bazı antitumor ilacların etkileri in vitro şartlarda esteraz aktivitesi metodu kullanılarak incelendi. İnhibitor etkisi gosteren ilacların IC50 değerleri hesaplandı. Enzimi ihhibe eden antitumor ilaclar icin Ki değerleri ve inhibisyon tipleri Lineweaver-Burk grafikleri ayrı ayrı cizilerek belirlendi. Bulgular: Fuzyon proteinin molekul ağırlığı yaklaşık olarak 85 kDa’du. Optimal IPTG indukleme konsantrasyonu ve buyume sıcaklığı 1,0 mM ve 30°C olarak bulundu. Fuzyon protein nikel-nitrilotriasetik asit (Ni-NTA) afinite kromatografisi kullanılarak yaklaşık olarak 43707 EUxmg-1 spesifik aktiviteyle; %92,58 verimle; 3,07 saflaştırma katsayısıyla saflaştırıldı. Sonuc: CA IX enzimi, kanser tanısında belirtec olarak klinik rol oynadığından ve CA IX enziminin spesifik inhibitorlerinin kullanımı kansere karşı mucadelede yararlı olacağından dolayı calışmamız onem arzetmektedir. Rekombinant hCA IX enzimi uzerine yapılacak olan in vitro inhibisyon calışmaları CA IX’un aşırı ekspresyon gosterdiği kanserler icin antikanser ilaclarının geliştirilmesine ışık tutabilir.

Comparison of inhibition effects of some benzoic acid derivatives on sheep heart carbonic anhydrase

Carbonic anhydrase (CA) is a family of metalloenzymes that requires Zn as a cofactor and catalyze the quick conversion of CO2 to HCO3− and H+. Inhibitors of the carbonic anhydrases (CAs) have medical usage of significant diseases such as glaucoma, epilepsy, gastroduodenal ulcers, acid-base disequilibria and neurological disorders. In the present study, inhibition of CA with some benzoic derivatives (1-6) were investigated. Sheep heart CA (shCA) enzyme was isolated by means of designed affinity chromatography gel (cellulose-benzyl-sulfanylamide) 42.45-fold in a yield of 44 % with 564.65 EU/mg. Purified shCA enzyme was used in vitro studies. In the studies, IC50 values were calculated for 3-aminobenzoic acid (1), 4-aminobenzoic acid (2), 2-hydroxybenzoic acid (3), 2-benzoylbenzoic acid (4), 2,3-dimethoxybenzoic acid (5), and 3,4,5-trimethoxybenzoic acid (6), showing the inhibition effects on the purified enzyme. Such molecules can be used as pioneer for discovery of novel effective CA inhibitors for medicin...