Ferencz Ágnes

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   Identification and expression of the genes of  Metal-Responsive Transcription Factor-1 and  glutathione peroxidases of common carp    Summery of Ph.D. Thesis      Ágnes Ferencz         Supervisor: Dr. Edit Hermesz        Ph.D. School of Environmental Sciences    University of Szeged  Faculty of Science and Informatics  Department of Biochemistry and Molecular Biology  2010.   1 1. Introduction  Environmental pollution, including the contamination of rivers and lakes, is still a persistent and significant problem. In some cases, it may be increased by disasters, such as the pollution of the River Tisza with cyanide and heavy metals in 2000. Fish are in great danger, because they accumulate harmful substances in their bodies via their food, and they are permanently exposed to dissolved substances through their gills and skins.  Almost all physiological processes are influenced by heavy metals. The proteins, nucleic acids are damaged and the formations of reactive oxygen species (ROS) are induced by metals, which may cause intensified cell and tissue damage. The rapid response to environmental stress is a general characteristic of organisms, which (from bacteria to mammals) have a high degree of homology in the response of the cells.  One of main pathways of stress responses is based on the metallothioneins (MTs) and their primary transcription regulator, metal-responsive transcription factor (MTF-1). Another pathway is associated with reduced glutathione (GSH) and certain enzymes, such as glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione synthetase (GSS). It is clear, therefore, that the mtf-1, gpx1 and gpx4 genes are key members in the antioxidant defence system. Study of the expression patterns of these genes contributes to a better understanding of the stress response.  Because of the high metal-binding capacities of MTs, they play important roles in the homeostasis of essential metal ions (Zn2+ and Cu2+) and in the elimination of toxic heavy metals (Cd2+, As3+ and Pb2+). The expression of MTs is primarily transcriptionally regulated and the Zn2+-sensitive MTF-1 is an essential participant in this process.  GSH is an antioxidant molecule, consisting only three amino acids (-Glu-Cys-Gly). In the event of oxidative stress, GSH is readily oxidized, because of the free SH group; when exposed to heavy metals, GSH binds metal ions. GSH also participates in the regeneration of ascorbic acid and vitamin E. GSH is a cofactor for antioxidant enzymes reducing H2O2 and lipid peroxides and neutralizing xenobiotics. The GPx enzymes protect against the harmful effects of ROS, because they reduce H2O2, some organic lipid peroxides and cholesterol esters, during the oxidation of GSH. The phospholipid hydroperoxide GPx (GPx4) binds directly to the membranes, and prevents the initiation of radical chain reactions. Eight different GPx families have been identified in mammals; in fish, only the classical GPx1 and GPx4 have been studied.  2 2. Aims    The main aims of our group are the identification and functional characterization of stress-response gene families in fish. At the beginning of these experiments, our primary aim was to identify gene(s) parts of defense system against heavy metal toxicity in the common carp, for example mtf-1, gpx1 and gpx4.   Other aims were to study the expressions of mtf-1 and gpx genes in the liver and the kidney, the organs most involved in the processing and excretion of toxic agents, and also in the heart, the muscle and three isolated brain regions (olfactory lobe, midbrain region and cerebellum) of untreated fish, and to follow the expressions of these genes in the liver and brain of stressed fish. The liver is one of the most important organs of detoxification, while the brain has high oxygen content, and is therefore the primary target of the harmful effects of ROS. The effects of treatment with a divalent heavy metal (Cd2+), a variable valence heavy metal (As3+; As5+) and physical stress (rapid temperature change) was investigated.   In the third part of the experiments, our aims were to examine the parameters of cell damage, such as lipid peroxidation, the changes in the amounts of reduced and oxidized GSH, the ratio of single and double-strand DNA, and the enzyme activities of GPx and GR after Cd2+ treatment.    3. Materials and methods   Animals and treatment conditions   Common carp (Cyprinus carpio L.), obtained from the Tisza Fish Farm, Szeged, were acclimatized under fasting conditions in well-aerated 400-l water tanks over a 3-week period. In cold shock experiments, the fish were transferred from 12 oC to 5 oC for 1 or 5 h and tissues were taken after a 1-h recovery period, following cold treatment at the acclimatization temperature. For metal treatment, the carp were transferred into 100-l water tanks containing 10 mg/l Cd2+ (Cd(CH3COO)2x2H2O) or 10 mg/l As3+ (Na2HAsO4x7H2O), under static conditions. Samples were obtained directly after the cold shock, or after the 1-h recovery period. At each time point of the treatments, 3 or 4 animals were used for the measurements.  3 Applied Methods  Preparation of nucleic acid (genomic DNA, mRNA, plasmid DNA)  In vitro recombinant DNA techniques: ligation, transformation Reverse transcription polymerase chain reaction  Determination of GSH / GSSG levels  Measurement of lipid peroxidation  Measurement of GPx and GR activity  Fluorimetric method for determination of the ratio of DNA breaks  Phylogenetic analysis    4. Results    In the common carp, as in most fish, sequence analysis indicated a single gpx1 gene, in contrast with the zebrafish (another member of the Cyprinidae), where two gpx1 genes were found. In higher vertebrates, gpx4 has been identified as a single copy gene with multiple transcription and translation start sites, but in the common carp, as in other fish species (zebrafish, goldfish and salmon), two gpx4, gpx4a and gpx4b, have been isolated. More than 95% of the protein coding regions has been determined in all three gpx genes of the carp.   Two different mtf-1 genes (mtf-1.1 and mtf-1.2) and the splice-variant of mtf-1.1 (mtf-1.1a) have been identified in the common carp. The overall identity of the deduced amino acid sequences of MTF-1.1 and MTF-1.2 was also 98%. The two sequences could be aligned with 1 gap and 9 amino acid differences, located in the C-terminal transcriptional activation domains, mostly in the proline-rich and serine/threonine-rich domains. The two MTF-1 proteins of the carp therefore probably interact with different partners. The common carp, like the other members of the Cyprinidae, demonstrates many genome and gene duplications; the common carp is a tetraploid species. In the case of the isoform, MTF-1.1a, the amino acid sequence of the C-terminal region of the original MTF-1.1 is altered by a frame shift. The DNA-binding domains of MTF-1.1a are identical to those of the MTF-1.1 protein, but the acidic transactivation domain is changed and the proline- and serine/threonine-rich domains are missing. MTF-1 without the transcriptional activation domains retains its translocating and DNA-binding capacity, but is presumably unable to activate its target genes.  4  The mRNA levels of gpx4a were expressed in all examined tissues of the carp under unstressed conditions; the highest level was found in the liver. A high expression of gpx4b was determined in the olfactory lobe, but in the kidney and cerebellum it was virtually undetected. The expressions of gpx1 were known in the liver, kidney and brain of the goldfish and silver carp, but the expression of the gpx1 gene of the carp differs significantly from those of other fish. The carp gpx1 was detected with the highest level in the heart, kidney and midbrain regions. In other fish, the gpx1 expression was liver-specific, and in the goldfish brain it was undetectable.   The tissue-specific expression of the mtf-1 gene was undetectable by Northern hybridization in untreated fish and mammal species. All three mtf-1 mRNAs of the common carp were detected in significant quantities in the brain. In the liver, the level of mtf-1.1 mRNA was low and the level of mtf-1.1a mRNA was at the limit of detection. The mtf-1.2 mRNA was virtually undetected in any organ but the brain. The mtf-1.1:mtf-1.2 ratio in the brain was  3:2. In all examined tissues, the expression of mtf-1.1 was the determining factor. The fish were treated with 10 mg / l Cd2+ and As3+ for 24-96 hours. The expressions of the gpx1, gpx4a and mtf-1.1 genes were slightly increased after Cd2+treatment in the liver, but Cd2+ did not influence the levels of mtf-1.2 and mtf-1.1a mRNA. The level of gpx4b mRNA was virtually undetected as early as after 24 h. In the brain regions, time-dependent decreases were measured in the expressions of all examined genes. The As3+ treatment caused lower changes, but with similar tendencies in both tissues, except in the case of mtf-1.1a. After 48 h of As3+ exposure, the mRNA levels of the spliced variant were increased in both the liver and the brain. There was a significant difference, in that the expression of the gpx1 gene was undetected after Cd2+treatment in these tissues of the goldfish. Rapid changes in water temperature were chosen as physical stress, because fish in a freshwater environment are subject to temperature fluctuations. Relatively rapid changes in water temperature can influence the gene expressions and induce an oxidative stress-like status in the tissues. The fish were exposed to a 1- or 5-h 7 °C temperature drop and some fish were then allowed a 1-h recovery period to the temperature of incubation, which is basically a heat shock for them. The expressions of the mtf-1 genes showed induction after the 5-h treatment, while those of the gpx1 genes were increased after the 1-h treatment. The gpx4a mRNA level was decreased in both tissues. The changes in the mtf-1, gpx1 and gpx4a genes were not influenced by the 1-h recovery. In the case of the expressions of the gpx4b gene, which were decreased by metal exposure, the direct exposure to low temperature increased  5 the transcription by ~ 2.5-fold, but it had returned to the control level by the end of the 1-h recovery period.  In addition to the expressions of the stress genes following high-dose Cd2+ treatment, other parameters of cell damage and the activities of some key antioxidant enzymes were investigated. The lipid peroxidation, the ratio of single- and double-stranded DNA and the changes in the reduced and oxidized GSH levels are the most frequently studied parameters of cell damage. In the early stages, damage to the cells was indicated by the changes in three parameters, but the repair systems were activated effectively after the 48-hour treatment. The activation of two enzymes of GSH homeostasis, the GPx and GR, was induced.  The identification and expression analyses of several genes were continued in our current work, which will promote a better understanding of the stress reactions of fish after exposure to environmental effects.    6 Publications  Publications directly used in and related to the thesis:  Agnes Ferencz and Edit Hermesz. Identification and characterization of two mtf-1 genes in common carp.  Comp. Biochem. and Physiol. Part C. 2008. 128(3), 457-465. IF2009: 2,582  Agnes Ferencz and Edit Hermesz. Identification of a splice variant of the metal responsive transcription factor mtf-1.  Comp. Biochem. and Physiol. Part C. 2009. 150, 113-117. IF2009: 2,582  Edit Hermesz and Agnes Ferencz. Identification of two phospholipid hydroperoxide gluthatione peroxidase (GPx4) genes in carp.  Comp. Biochem. and Physiol. Part C. 2009. 150, 101-106. IF2009: 2,582  Other publications:  K. Said Ali, Agnes Ferencz, Aranka K. Deér, J. Nemcsók and Edit Hermesz. Expression of two metallothionein genes in different brain regions of common carp. Acta Biol. Hung. 2009. 60(2), 149-158.  IF2009: 0,551  Zsuzsanna Hracsko, Edit Hermesz, Agnes Ferencz, Hajnalka Orvos, Z. Novak, A. Pal, Ilona S. Varga. Endothelial nitric oxide synthase is up-regulated in the umbilical cord in pregnancies complicated with intrauterine growth retardation. In Vivo. 2009. 23(5), 727-732. IF2009:1,171   7 K. Said Ali, Agnes Ferencz, J. Nemcsók, Edit Hermesz. Expressions of heat shock and metallothionein genes in the heart of common carp (Cyprinus carpio): effects of temperature shock and heavy metal exposure.  Acta Biol. Hung. 2010. 61(1), 10-23. IF2009: 0,551  Conference participations and posters:  K. S. Ali, Agnes Ferencz, Magdolna Abraham, Edit Hermesz. Induction of heat shock protein by heavy metal exposure and temperature stress in fish.  30th FEBS Congress, Budapest, Hungary, G1-018P. FEBS J. 2005. 272(1), 352-353.    IF2009: 3,042  Edit Hermesz, Agnes Ferencz, Aranka K. Deer, K. S. Ali. Isoform-specific stress response of two metallothionein genes in the heart of common carp during exposure to metal ions.  32nd FEBS Congress, Vienna, Austria, C4-94. FEBS J. 2007. 274(1), 362. IF2009: 3,042  Edit Hermesz, Sz. Abraham, Agnes Ferencz, Veronika Kovacs, M. Boros, G. Lazar Jr. Expression of metallothionein and heme oxygenase in gadolinium chloride-pretreated endotoxemic rats.  32nd FEBS Congress, Vienna, Austria, C4-95. FEBS J. 2007. 274(1), 362. IF2009: 3,042  S. Abraham, Agnes Ferencz, Andrea Szabo, G. Lazar, M. Boros, Edit Hermesz, G. Lazar Jr. Hepatic expression and regulation of metallothionein and heme oxygenase genes in endotoxemic rats with obstructive jaundice – the role of Kupffer cell.  42nd Congress of the European Society for Surgical Research (ESSR), Rotterdam, NL. European Surgical Res. 2007. 39(1), 38 – 39.  IF2009: 1,5  8 Agnes Ferencz, Edit Hermesz. Isolation and tissue specific expression of the mtf-1 gene of common carp (Cyprinus carpio).  33rd FEBS Congress and 11th IUBMB Conference, Athen, Greece, PP2C-8. FEBS J. 2008. 275(1), 146.  IF2009: 3,042   S. Abraham, Edit Hermesz, Agnes Ferencz, Andrea Szabo, Gy. Lazár, M. Boros, Gy. Lazar jr. Antioxidant defense system in endotoxemic rats with obstructive jaundice. 6th Congress of the international Federation of Shock Societies & 31St Annual Conference on Shock 7th International Conference on Complexity in Acute Illness, Cologne, Germany, P248. Shock 2008, 29(1), 76.  IF2009: 2,871  Agnes Ferencz, Edit Hermesz. Alternative splicing of Metal-responsive Transcription Factor (MTF-1).  34th FEBS Congress and Young Scientist Forum, Prague, Czech Republic, YSF24.  FEBS J. 2009. 276(1), 364-365. IF2009: 3,042  Edit Hermesz, Agnes Ferencz. Regulation of metal responsive transcription factor MTF-1 expression in common carp.  A Magyar Szabadgyök-Kutató Társaság V. Kongresszusa, Szeged, P-13. Acta Biol. Szegediensis. 2009. 53(1), 47.  Sz. Ábrahám, Edit Hermesz, Ágnes Ferencz, Andrea Szabó, Gy. Lázár, Gy. Lázár Jr. A metallothionein és hemoxigenáz gének expressziója és szabályozása májban, epeúti elzáródást követő endotoxémiában – a Kupffer sejtek szerepe. Magyar Sebészet. 2009. 62(3), 152.    

A Metal-responsive transcription factor-1 és a glutation peroxidáz család tagjait kódoló gének azonosítása és expressziójának jellemzése pontyban

http://doktori.bibl.u-szeged.hu/929/1/Dolgozat_Ferencz_Agnes.pdf

A Metal-responsive transcription factor-1 és a glutation peroxidáz család tagjait kódoló gének azonosítása és expressziójának jellemzése pontyban

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