Characterization of cytosolic glutathione peroxidase and phospholipid-hydroperoxide glutathione peroxidase genes in rainbow trout (Oncorhynchus mykiss) and their modulation by in vitro selenium exposure

D. Pacitti, T. Wang, M. M. Page, S. A. M. Martin, J. Sweetman, J. Feldmann, C. J. Secombes

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Selenium (Se) is an oligonutrient with both essential biological functions and recognized harmful effects. As the selenocysteine (SeCys) amino acid, selenium is integrated in several Se-containing proteins (selenoproteins), many of which are fundamental for cell homeostasis. Nevertheless, selenium may exert toxic effects at levels marginally above those required, mainly through the generation of reactive oxygen species (ROS). The selenium chemical speciation can strongly affect the bioavailability of this metal and its impact on metabolism, dictating the levels that can be beneficial or detrimental towards an organism. Glutathione peroxidase (GPxs) is the largest and the most studied selenoprotein family. Cytosolic glutathione peroxidase (cGPx, GPx1) and phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) are widely distributed throughout tissues, and play a pivotal role in regulating the oxidative status in the cell. In this study we have cloned GPx1 and GPx4 genes in rainbow trout (Oncorhynchus mykiss). The constitutive mRNA expression of these GPx genes was examined in 18 trout tissues and their responsiveness to Se availability was analysed using a rainbow trout liver cell line (RTL). An inorganic (sodium selenite, Na2SeO3) and organic (selenocysteine, Cys-Se-Se-Cys) selenocompound have been used as Se sources. GPx1 activity was also tested to verify the impact of transcript changes on the enzymatic function of these molecules. To understand if the results obtained from the transcript expression analysis were due to Se bioavailability or generation of ROS, the cytoxicity of the two selenocompounds was tested by measuring the impact of Se on cell membrane integrity. Lastly, Se availability was quantified by mass spectrophotometry to determine the amount of Se in the cell culture media, the Se background due to the foetal calf serum supplement and the contribution from the two selenocompounds used in the treatments. Three isoforms of genes for both GPx1 (GPx1a, 1b1 and 1b2) and GPx4 (GPx4a1, a2 and b) have been identified. The discovery of a third gene encoding for GPx1 and GPx4 hints that salmonids may have the biggest selenoproteome amongst all vertebrates. Transcripts of GPx4 genes were more highly expressed in most tissues examined in vivo (except blood, head kidney and spleen), whereas those of the GPx1 genes were more responsive to selenium exposure in vitro, especially to the organic form. Interestingly, GPx1a was the most sensitive to selenium availability in non stressful conditions, whereas GPx1b1 and GPx1b2 were highly induced by exposure to selenium levels that had some toxic effects on the cells. Although the different concentrations tested of the two selenocompounds modulate GPx1 transcript expression to various degrees, no significant change of GPx1 enzymatic activity was detectable. Our results lead us to conclude that trout GPx1 transcripts expression level may represent a sensitive biomarker for selenium intake, helping to evaluate if selenium concentration and chemical speciation impact on cell homeostasis.
Original languageEnglish
Pages (from-to)97-111
Number of pages15
JournalAquatic Toxicology
Volume130-131
DOIs
Publication statusPublished - 15 Apr 2013

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phospholipid-hydroperoxide glutathione peroxidase
Oncorhynchus mykiss
Selenium
Glutathione Peroxidase
phospholipid
selenium
glutathione peroxidase
rainbow
gene
Genes
genes
Selenoproteins
Selenocysteine
selenocysteine
In Vitro Techniques
exposure
selenoproteins
chemical speciation
speciation (chemistry)
Trout

Keywords

  • amino acid sequence
  • animals
  • base sequence
  • cell line
  • cloning, molecular
  • DNA, complementary
  • fish proteins
  • glutathione peroxidase
  • mass spectrometry
  • molecular sequence data
  • Oncorhynchus mykiss
  • organ specificity
  • phylogeny
  • polymerase chain reaction
  • RNA, messenger
  • selenocysteine
  • sequence alignment
  • sodium selenite
  • selenium
  • salmonids
  • RTL cell line

Cite this

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title = "Characterization of cytosolic glutathione peroxidase and phospholipid-hydroperoxide glutathione peroxidase genes in rainbow trout (Oncorhynchus mykiss) and their modulation by in vitro selenium exposure",
abstract = "Selenium (Se) is an oligonutrient with both essential biological functions and recognized harmful effects. As the selenocysteine (SeCys) amino acid, selenium is integrated in several Se-containing proteins (selenoproteins), many of which are fundamental for cell homeostasis. Nevertheless, selenium may exert toxic effects at levels marginally above those required, mainly through the generation of reactive oxygen species (ROS). The selenium chemical speciation can strongly affect the bioavailability of this metal and its impact on metabolism, dictating the levels that can be beneficial or detrimental towards an organism. Glutathione peroxidase (GPxs) is the largest and the most studied selenoprotein family. Cytosolic glutathione peroxidase (cGPx, GPx1) and phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) are widely distributed throughout tissues, and play a pivotal role in regulating the oxidative status in the cell. In this study we have cloned GPx1 and GPx4 genes in rainbow trout (Oncorhynchus mykiss). The constitutive mRNA expression of these GPx genes was examined in 18 trout tissues and their responsiveness to Se availability was analysed using a rainbow trout liver cell line (RTL). An inorganic (sodium selenite, Na2SeO3) and organic (selenocysteine, Cys-Se-Se-Cys) selenocompound have been used as Se sources. GPx1 activity was also tested to verify the impact of transcript changes on the enzymatic function of these molecules. To understand if the results obtained from the transcript expression analysis were due to Se bioavailability or generation of ROS, the cytoxicity of the two selenocompounds was tested by measuring the impact of Se on cell membrane integrity. Lastly, Se availability was quantified by mass spectrophotometry to determine the amount of Se in the cell culture media, the Se background due to the foetal calf serum supplement and the contribution from the two selenocompounds used in the treatments. Three isoforms of genes for both GPx1 (GPx1a, 1b1 and 1b2) and GPx4 (GPx4a1, a2 and b) have been identified. The discovery of a third gene encoding for GPx1 and GPx4 hints that salmonids may have the biggest selenoproteome amongst all vertebrates. Transcripts of GPx4 genes were more highly expressed in most tissues examined in vivo (except blood, head kidney and spleen), whereas those of the GPx1 genes were more responsive to selenium exposure in vitro, especially to the organic form. Interestingly, GPx1a was the most sensitive to selenium availability in non stressful conditions, whereas GPx1b1 and GPx1b2 were highly induced by exposure to selenium levels that had some toxic effects on the cells. Although the different concentrations tested of the two selenocompounds modulate GPx1 transcript expression to various degrees, no significant change of GPx1 enzymatic activity was detectable. Our results lead us to conclude that trout GPx1 transcripts expression level may represent a sensitive biomarker for selenium intake, helping to evaluate if selenium concentration and chemical speciation impact on cell homeostasis.",
keywords = "amino acid sequence, animals, base sequence, cell line, cloning, molecular, DNA, complementary, fish proteins, glutathione peroxidase, mass spectrometry, molecular sequence data, Oncorhynchus mykiss, organ specificity, phylogeny, polymerase chain reaction, RNA, messenger, selenocysteine, sequence alignment, sodium selenite, selenium, salmonids, RTL cell line",
author = "D. Pacitti and T. Wang and Page, {M. M.} and Martin, {S. A. M.} and J. Sweetman and J. Feldmann and Secombes, {C. J.}",
year = "2013",
month = "4",
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language = "English",
volume = "130-131",
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TY - JOUR

T1 - Characterization of cytosolic glutathione peroxidase and phospholipid-hydroperoxide glutathione peroxidase genes in rainbow trout (Oncorhynchus mykiss) and their modulation by in vitro selenium exposure

AU - Pacitti, D.

AU - Wang, T.

AU - Page, M. M.

AU - Martin, S. A. M.

AU - Sweetman, J.

AU - Feldmann, J.

AU - Secombes, C. J.

PY - 2013/4/15

Y1 - 2013/4/15

N2 - Selenium (Se) is an oligonutrient with both essential biological functions and recognized harmful effects. As the selenocysteine (SeCys) amino acid, selenium is integrated in several Se-containing proteins (selenoproteins), many of which are fundamental for cell homeostasis. Nevertheless, selenium may exert toxic effects at levels marginally above those required, mainly through the generation of reactive oxygen species (ROS). The selenium chemical speciation can strongly affect the bioavailability of this metal and its impact on metabolism, dictating the levels that can be beneficial or detrimental towards an organism. Glutathione peroxidase (GPxs) is the largest and the most studied selenoprotein family. Cytosolic glutathione peroxidase (cGPx, GPx1) and phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) are widely distributed throughout tissues, and play a pivotal role in regulating the oxidative status in the cell. In this study we have cloned GPx1 and GPx4 genes in rainbow trout (Oncorhynchus mykiss). The constitutive mRNA expression of these GPx genes was examined in 18 trout tissues and their responsiveness to Se availability was analysed using a rainbow trout liver cell line (RTL). An inorganic (sodium selenite, Na2SeO3) and organic (selenocysteine, Cys-Se-Se-Cys) selenocompound have been used as Se sources. GPx1 activity was also tested to verify the impact of transcript changes on the enzymatic function of these molecules. To understand if the results obtained from the transcript expression analysis were due to Se bioavailability or generation of ROS, the cytoxicity of the two selenocompounds was tested by measuring the impact of Se on cell membrane integrity. Lastly, Se availability was quantified by mass spectrophotometry to determine the amount of Se in the cell culture media, the Se background due to the foetal calf serum supplement and the contribution from the two selenocompounds used in the treatments. Three isoforms of genes for both GPx1 (GPx1a, 1b1 and 1b2) and GPx4 (GPx4a1, a2 and b) have been identified. The discovery of a third gene encoding for GPx1 and GPx4 hints that salmonids may have the biggest selenoproteome amongst all vertebrates. Transcripts of GPx4 genes were more highly expressed in most tissues examined in vivo (except blood, head kidney and spleen), whereas those of the GPx1 genes were more responsive to selenium exposure in vitro, especially to the organic form. Interestingly, GPx1a was the most sensitive to selenium availability in non stressful conditions, whereas GPx1b1 and GPx1b2 were highly induced by exposure to selenium levels that had some toxic effects on the cells. Although the different concentrations tested of the two selenocompounds modulate GPx1 transcript expression to various degrees, no significant change of GPx1 enzymatic activity was detectable. Our results lead us to conclude that trout GPx1 transcripts expression level may represent a sensitive biomarker for selenium intake, helping to evaluate if selenium concentration and chemical speciation impact on cell homeostasis.

AB - Selenium (Se) is an oligonutrient with both essential biological functions and recognized harmful effects. As the selenocysteine (SeCys) amino acid, selenium is integrated in several Se-containing proteins (selenoproteins), many of which are fundamental for cell homeostasis. Nevertheless, selenium may exert toxic effects at levels marginally above those required, mainly through the generation of reactive oxygen species (ROS). The selenium chemical speciation can strongly affect the bioavailability of this metal and its impact on metabolism, dictating the levels that can be beneficial or detrimental towards an organism. Glutathione peroxidase (GPxs) is the largest and the most studied selenoprotein family. Cytosolic glutathione peroxidase (cGPx, GPx1) and phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) are widely distributed throughout tissues, and play a pivotal role in regulating the oxidative status in the cell. In this study we have cloned GPx1 and GPx4 genes in rainbow trout (Oncorhynchus mykiss). The constitutive mRNA expression of these GPx genes was examined in 18 trout tissues and their responsiveness to Se availability was analysed using a rainbow trout liver cell line (RTL). An inorganic (sodium selenite, Na2SeO3) and organic (selenocysteine, Cys-Se-Se-Cys) selenocompound have been used as Se sources. GPx1 activity was also tested to verify the impact of transcript changes on the enzymatic function of these molecules. To understand if the results obtained from the transcript expression analysis were due to Se bioavailability or generation of ROS, the cytoxicity of the two selenocompounds was tested by measuring the impact of Se on cell membrane integrity. Lastly, Se availability was quantified by mass spectrophotometry to determine the amount of Se in the cell culture media, the Se background due to the foetal calf serum supplement and the contribution from the two selenocompounds used in the treatments. Three isoforms of genes for both GPx1 (GPx1a, 1b1 and 1b2) and GPx4 (GPx4a1, a2 and b) have been identified. The discovery of a third gene encoding for GPx1 and GPx4 hints that salmonids may have the biggest selenoproteome amongst all vertebrates. Transcripts of GPx4 genes were more highly expressed in most tissues examined in vivo (except blood, head kidney and spleen), whereas those of the GPx1 genes were more responsive to selenium exposure in vitro, especially to the organic form. Interestingly, GPx1a was the most sensitive to selenium availability in non stressful conditions, whereas GPx1b1 and GPx1b2 were highly induced by exposure to selenium levels that had some toxic effects on the cells. Although the different concentrations tested of the two selenocompounds modulate GPx1 transcript expression to various degrees, no significant change of GPx1 enzymatic activity was detectable. Our results lead us to conclude that trout GPx1 transcripts expression level may represent a sensitive biomarker for selenium intake, helping to evaluate if selenium concentration and chemical speciation impact on cell homeostasis.

KW - amino acid sequence

KW - animals

KW - base sequence

KW - cell line

KW - cloning, molecular

KW - DNA, complementary

KW - fish proteins

KW - glutathione peroxidase

KW - mass spectrometry

KW - molecular sequence data

KW - Oncorhynchus mykiss

KW - organ specificity

KW - phylogeny

KW - polymerase chain reaction

KW - RNA, messenger

KW - selenocysteine

KW - sequence alignment

KW - sodium selenite

KW - selenium

KW - salmonids

KW - RTL cell line

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U2 - 10.1016/j.aquatox.2012.12.020

DO - 10.1016/j.aquatox.2012.12.020

M3 - Article

VL - 130-131

SP - 97

EP - 111

JO - Aquatic Toxicology

JF - Aquatic Toxicology

SN - 0166-445X

ER -