Alaattin Sen

Determination of 7-ethoxyresorufin-o-deethylase (EROD) induction in leaping mullet (Liza saliens) from the highly contaminated Aliaga Bay, Turkey

Pollution of the aquatic environment is a global concern owing to the devastating effects of contaminants whose levels are growing at an alarming rate, and it has become a major threat for marine organisms, as well as to humans as consumers. This study has been carried out on leaping mullet obtained from Aliaga Bay, which is located on the west coast of the Aegean Sea near Izmir and hosts the world’s fifth largest shipyard, plus a broad range of industrial activities, including an oil refinery and a paper factory. The waste from these industries, combined with municipal sewer discharges, is the main cause of pollution in this region. There is no national documentation or research on the determination of pollution resulting from the industrial activities in this area. In the present study, the degree of induction of CYP4501A-associated 7-ethoxyresorufin O-deethylase (EROD) activity and immunochemical detection of CYP1A1 in the liver of leaping mullet (Lisa saliens) were used as biomarkers for the assessment of polycyclic aromatic hydrocarbon (PAH)-type organic pollutants in Aliaga Bay. Mullet caught from different locations of the bay had approximately 52 times more EROD activity than the feral fish sampled from a clean reference site near Foca, Izmir. The results of this study indicate that Aliaga Bay is highly contaminated with PAH-type organic pollutants.

A Comparative Study for the Evaluation of Two Doses of Ellagic Acid on Hepatic Drug Metabolizing and Antioxidant Enzymes in the Rat

The present study was designed to evaluate different doses of ellagic acid (EA) in vivo in rats for its potential to modulate hepatic phases I, II, and antioxidant enzymes. EA (10 or 30 mg/kg/day, intragastrically) was administered for 14 consecutive days, and activity, protein, and mRNA levels were determined. Although the cytochrome P450 (CYP) 2B and CYP2E enzyme activities were decreased significantly, the activities of all other enzymes were unchanged with the 10 mg/kg/day EA. In addition, western-blot and qRT-PCR results clearly corroborated the above enzyme expressions. On the other hand, while the NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were increased significantly, CYP1A, 2B, 2C, 2E, and 19 enzyme activities were reduced significantly with 30 mg/kg/day EA. In addition, CYP2B, 2C6, 2E1, and 19 protein and mRNA levels were substantially decreased by the 30 mg/kg/day dose of EA, but the CYP1A protein, and mRNA levels were not changed. CYP3A enzyme activity, protein and mRNA levels were not altered by neither 10 nor 30 mg/kg/day ellagic acid. These results indicate that EA exerts a dose-dependent impact on the metabolism of chemical carcinogens and drugs by affecting the enzymes involved in xenobiotics activation/detoxification and antioxidant pathways.

In vivo effects of Urtica urens (dwarf nettle) on the expression of CYP1A in control and 3-methylcholanthrene-exposed rats

The in vivo effects of the intraperitoneal administration of an Urtica urens L. (dwarf nettle) seed extract were examined on the hepatic, pulmonary, and renal cytochrome P450-dependent monooxygenase activities of rats co-administered with 3-methylcholanthrene (MC). Urtica extract was administered by intraperitoneal injection to male Wistar rats at 200mgkg−1day−1 for 4 days from which were also co-administered with intraperitoneal injection of 50mg of MCkg−1 of body weight twice on days 1 and 3. MC treatment increased the 7-ethoxyresorufin O-deethylase (EROD) activity of the liver, lung, and kidney 54-, 21-, and 119-fold, respectively. Urtica treatment substantially reduced the 3MC induction of hepatic, lung, and renal EROD activity by 79, 42, and 50%, respectively. Similarly, compared with the control, MROD activities in liver and kidney were increased after MC administration, and these increases were significantly inhibited by Urtica. reverse transcriptase-polymerase chain reaction (RT-PCR) analysis clearly showed that the hepatic CYP1A1 and CYP1A2 mRNA levels substantially increased after treatment with MC, which was suppressed by Urtica supplementation. Western blotting studies also supported the alterations observed in the catalytic activities and mRNA levels. In conclusion, substantial reduction in CYP1A1 and CYP1A2 expression levels and related activities with Urtica are possibly associated with a potential chemoprotective ability of the Urtica due to the anticipated decrease in the activation of environmental chemical carcinogens through modulation of the CYP1A enzymes.

Drug interaction potential of the seed extract of Urtica urens L. (dwarf Nettle)

Dwarf nettle (Urtica urens) seed extract was examined in vivo in the rat for its potential to modulate drug metabolizing enzymes including aminopyrine N‐demethylase (APND; CYP2C6), aniline 4‐hydroxylase (A4H; CYP2E1), nitrosodimethylamine N‐demethylase (NDMA‐ND; CYP2E1) erythromycin N‐demethylase (ERND; CYP3A1) CYP2D1/2 and glutathione S‐transferase (GST). RT‐PCR data and western blotting studies clearly demonstrated that CYP2C6 and CYP2E1 mRNA levels were substantially increased after Urtica treatment, while the level of CYP3A1 mRNA decreased and that of CYP2D1/2 remained unchanged. Urtica treatment significantly induced GST activity in the liver, lung and kidney (66‐, 46‐ and 31‐fold, respectively) while decreasing that of APND (35‐, 61‐ and 94‐fold) and NDMA‐ND (23, 28 and 54‐fold). ERND activity in liver was reduced 45‐fold, but increased in the lung and kidney (78‐ and 144‐fold) after Urtica treatment. These results indicate that Urtica seed extract may have the potential to inhibit and/or induce the metabolism of certain co‐administered drugs. Copyright

Epilobium hirsutum alters xenobiotic metabolizing CYP1A1, CYP2E1, NQO1 and GPx activities, mRNA and protein levels in rats.

Abstract Context: Natural products have attracted increasing interests due to their use in flavoring, nutrition, cosmetics, pharmacy and medicine. Epilobium hirsutum L. (Onagraceae) is known for its analgesic, antimicrobial, and antiproliferative activity. CYP1A1 and CYP2E1, xenobiotic metabolizing enzymes, serve as a metabolic activation route yielding reactive metabolites that are eliminated by the action of NQO1 and glutathione peroxidase (GPx) enzymes. Objective: This study investigated in vivo effects of Epilobium hirsutum (EH) on CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA expressions in liver. Materials and methods: Male Wistar Albino rats were injected with EH at a dose of 37.5 mg/kg i.p. daily for 9 d. CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA levels were determined by enzyme assays, Western blotting and qPCR, respectively. Results: CYP1A1 associated ethoxyresorufin-O-deethylase activity of control and EH-treated animals were found as 6.54 ± 1.21 and 4.48 ± 1.67 nmol/min/mg, respectively. CYP2E1 associated aniline 4-hydroxylase of control and EH group were 0.537 ± 0.011 and 0.109 ± 0.01 nmol/min/mg, respectively. However, EH treatment increased the GPx and NQO1 activities from 0.069 ± 0.015 to 0.107 ± 0.026 nmol/min/mg and from 163.34 ± 92 to 588.3 ± 14 nmol/min/mg, respectively. Furthermore, protein and mRNA expression analysis revealed that CYP1A1 and CYP2E1 levels were decreased while those of NQO1 and GPx increased after EH treatment. Discussion and conclusion: Our current data suggest that the metabolism of xenobiotics, including drugs, may be altered due to changes in the expression and activity of these proteins by EH.

Alteration of drug metabolizing enzymes in sulphite oxidase deficiency

The aim of this study was to investigate the possible effects of sulphite oxidase (SOX, E.C. 1.8.3.1) deficiency on xenobiotic metabolism. For this purpose, SOX deficiency was produced in rats by the administration of a low molybdenum diet with concurrent addition of 200 ppm tungsten to their drinking water. First, hepatic SOX activity in deficient groups was measured to confirm SOX deficiency. Then, aminopyrine N-demethylase, aniline 4-hydroxylase, aromatase, caffeine N-demethylase, cytochrome b5 reductase, erythromycin N-demethylase, ethoxyresorufin O-deethylase, glutathione S-transferase, N-nitrosodimethylamine N-demethylase and penthoxyresorufin O-deethylase activities were determined to follow changes in the activity of drug metabolizing enzymes in SOX-deficient rats. Our results clearly demonstrated that SOX deficiency significantly elevated A4H, caffeine N-demethylase, erythromycin N-demethylase and N-nitrosodimethylamine N-demethylase activities while decreasing ethoxyresorufin O-deethylase and aromatase activities. These alterations in drug metabolizing enzymes can contribute to the varying susceptibility and response of sulphite-sensitive individuals to different drugs and/or therapeutics used for treatments.

Contribution of ellagic acid on the antioxidant potential of medicinal plant Epilobium hirsutum

abstract In the present study, the possible role of ellagic acid (EA) on antioxidant potential of Epilobium hirsutum (EH) in rat liver was investigated. Wistar rats were intraperitoneally treated with 37.5 mg/kg of EH and 10 mg/kg of EA for 9 days. Effects of EH and EA on antioxidant [glutathione peroxidase (GPx) and superoxide dismutases (SOD)] and Phase II [NADPH quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTs)] enzyme activities, as well as protein and mRNA expressions of those, were investigated. Polyphenolic content of EH was determined by LC-MS/MS analysis. EH and EA injection to rats resulted in a significant increase of NQO1 (3.6-fold and 4.7-fold), GPx (1.45-fold), and SOD (1.34-fold and 1.27-fold) enzyme activities, whereas total GST (46% and 57%) and its isoforms,and GST mu (57% and 72%), and GST theta (60% and 68%) activities were significantly decreased. Western-blot and qRT-PCR analysis showed that NQO1 and GPx protein and mRNA expressions were increased significantly (P < 0.0001), whereas GST mu and GST theta were significantly decreased (P < 0.0001).

Association of the CYP1A1*2A, GSTT1 null, GSTM1 null, mEPHX*3, and XRCC1-399 genetic polymorphisms with ulcerative colitis.

Dear Editor: Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) that affects the large intestine and rectum. IBDs are believed to result from the interplay between a number of disease genes and environmental factors. Epidemiological data provide evidence that genetic predisposition to ulcerative colitis depends on the contribution of multiple genes rather than a single genetic factor. Recently, the number of ulcerative colitis-associated loci and genes, such as ECM1, IL23R, IL12B, HLA, NKX2-3, and VDR, has increased. However, mutations within these genes were absent in the majority of patients, suggesting the presence of additional susceptibility genes. It is suggested that reactive molecules, such as reactive oxygen species ROS, may play a pivotal role in the mechanism of inflammation by altering the intestinal permeability, which leads to tissue injury in the mucosa, ultimately resulting in tissue damage. Reactive molecules may be products of the endogenous metabolism of xenobiotics catalyzed by biotransformation enzymes. Changes in detoxification of xenobiotics that cause epithelial damage may confer susceptibility to UC. Chronic inflammation of the colon may further lead to carcinogenesis and tumor formation. Hence, polymorphic enzymes involved in the detoxification processes may be risk factors of UC and colon cancer. Biotransformation enzymes such as cytochrome P450s and GSTs perform vital roles in the metabolism of xenobiotics and ROS. They often convert parent compounds into highly reactive metabolites, such as epoxides, that may bind to cellular components and induce tissue damage and carcinogenesis. Detoxification of these reactive metabolites is carried out by either conjugating with glutathione, which is catalyzed by GSTs, or by hydration, which is catalyzed by epoxide hydrolases. X-ray repair cross-complementing groups (XRCCs) are important proteins of the DNA repair pathways. The XRCC1 gene is responsible for a scaffolding protein that directly associates with the processes of base excision repair or single-strand break repair. Numerous polymorphisms that result in altered enzyme activities have been described in the genes of these enzymes. It has been suggested that individual variations in the susceptibility to the mutagenic and carcinogenic activity of xenobiotics may be partially explained by differences in their activation and deactivation pathways. As chemical and oxidative stress may be involved in the etiology of ulcerative colitis, polymorphic genes encoding for these biotransformation enzymes may modulate the genetic susceptibility to ulcerative colitis and carcinogenesis in the colon. Therefore, this study was undertaken to determine whether genetic polymorphisms in biotransformation enzymes in patients with ulcerative colitis differ from those in healthy controls. Polymorphisms in genes encoding cytochrome P450 1A1 (CYP1A1), glutathione S-transferase mu-1 (GSTM1) and theta-1 (GSTT1), microsomal epoxide hydrolase (EPXH) and X-ray repair complementing group 1 (XRCC1) were investigated. The present case–control study was performed in subjects recruited between 2008 and 2010. A total of 161 consecutive patients with ulcerative colitis (94 males, 67 females; all Caucasian) consulting the outpatient clinic of the Department O. Buyukgoze : S. Arslan :A. Sen (*) Biology Department, Faculty of Arts & Sciences, Pamukkale University, Kinikli Campus, Kinikli, 20070, Denizli, Turkey e-mail: sena@pau.edu.tr

Potential use of the oligochaete Limnodrilus profundicola V., as a bioindicator of contaminant exposure.

Cholinesterase (ChE) and ethoxyresorufin‐O‐deethylase (EROD) were of special interest to this study as these biochemical tools have been widely used for the determination of exposure to pollutants. In this study, the freshwater oligochaete Limnodrilus profundicola was tested for its potential as a bioindicator of freshwater pollution. For this purpose, the ChE and EROD activities of L. profundicola and the level of polycyclic aromatic hydrocarbons (PAH) of water samples collected from different sites along the Curuksu stream on the Menderes River (the ancient Meander) running through south‐western Turkey were studied. First, these activities were characterized using, as model substrates, acetylthiocholine (ATC), propionylthiocholine (PTC), and butyrylthiocholine (BTC). Then, the in vivo effects of insecticides and pollutants on these activities were investigated. L. profundicola were exposed to various doses of methyl‐parathion, methomyl, and deltamethrin. Although significant inhibition of ChE was detected with each of the insecticides, the highest level of inhibition was observed with methyl‐parathion. In addition to the inhibition of ChE, the activity of EROD was induced by exposure to oil‐contaminated sediments. Thus, although L. profundicola has a reputation for being very resistant to pollution (although it is not insensitive to it), we demonstrated that it may potentially be used as a bioindicator species for contaminant exposure when ChE and EROD are used as biomarkers.

The isolation of tetrangomycin from terrestrial Streptomyces sp. CAH29: evaluation of antioxidant, anticancer, and anti-MRSA activity

A rhizosphere isolate Streptomyces sp. CAH29 was found to possess potent antibacterial and antifungal activity against a variety of test organisms. Based on 16S ribosomal ribonucleic acid sequence homology studies, this strain was found to be similar to Streptomyces stramineus (gene sequence similarity 99 %). The major bioactive metabolite produced by Streptomyces sp. CAH29 isolate was extracted, purified andidentified by nuclear magnetic resonance as tetrangomycin. This known anthraquinone-exhibited antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, methicillin resistant Staphylococcus aureus and Candida albicans with inhibition zones of 14, 10, 12 and 8 mm, respectively. Docking results demonstrate that tetrangomycin has a similar mode of action and a comparable docking score to bind to the dehydrosqualene synthase (CrtM) enzyme of methicillin resistant Staphylococcus aureus compared to the current inhibitor. Hence, this suggests that tetrangomycin has a potential to be used as an anti-methicillin resistant Staphylococcus aureus agent. Tetrangomycin also showed moderate free radical scavenging activity with 1,1-diphenyl-2-picryl-hydrazil. Tetrangomycin apparently decreased all of the studied cytokine (pro-inflammatory: interleukin 1B, interleukin 2, tumor necrosis factor and interleukin L6 and anti-inflammatory: interleukin 10) expression levels at IC50 concentrations in A459 (adenocarcinomic human alveolar basal epithelial) and LNCAP (human prostate adenocarcinoma) cell lines. In addition, it reduced Caspase 8 and 3 mRNA levels in LNCAP and A549 cells. This study describes for the first time novel in vitro immunosuppressive function of tetrangomycin by reducing the transcription of cytokine genes.