Induction of thioltransferase and thioredoxin/thioredoxin reductase systems in cultured porcine lenses under oxidative stress

Sungchur Moon, M Rohan Fernando, Marjorie F. Lou

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

PURPOSE. In view of the important antioxidant roles of thioltransferase (TTase), thioredoxin (Trx), and thioredoxin reductase (TR) in the lens, the present study was conducted to investigate the induction of these cytosolic enzymes in response to H2O2 stress in cultured lenses. METHODS. Porcine lenses were cultured, exposed to H2O2 for various lengths of time between 0 and 24 hours, and photographed to detect morphologic changes. The lenses were then harvested; dissected into epithelial layer, cortex, and nucleus; and homogenized for the determination of the glutathione (GSH) level. Pooled epithelial layers were used to examine TTase, Trx, and TR protein or mRNA levels. RESULTS. Treatment of lenses with H 2O2 caused distinct morphologic changes. Lower concentrations of H2O2 (0.2 mM) caused the lens to be hazy after 6 hours and to worsen progressively between 12 and 24 hours. Higher levels of H2O2 (0.5 mM) induced similar morphologic changes, but sooner (within 1 hour) and more severe. Both H2O 2-treated groups showed a dramatic and gradual GSH depletion during the 24-hour incubation, but the GSH level at 50% or above appeared to be essential in maintaining lens clarity. However, TTase, Trx, and TR activities, protein expressions, and mRNA transcriptions in the epithelial layers of these lenses were increased, but each enzyme had a distinct pattern. Under mild H 2O2 stress, a slow and transient activation of TTase, Trx, and TR was observed. However, under stronger H2O2 stress, all three enzymes showed a very rapid increase and then a steady decline in activity. Western blot and RT-PCR analyses revealed that this increase in activity in all three enzymes was due to the induction of protein and mRNA expression. In the control group (no oxidative stress) all three enzyme activities and their respective expressions remained constant throughout the experimental period. CONCLUSIONS. The data show that TTase, Trx, and TR activity and expression are induced in lens cells under oxidative stress, probably to protect and maintain the health of the lens.

Original languageEnglish (US)
Pages (from-to)3783-3789
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume46
Issue number10
DOIs
StatePublished - Oct 1 2005

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Glutaredoxins
Thioredoxin-Disulfide Reductase
Thioredoxins
Lenses
Oxidative Stress
Swine
Enzymes
Messenger RNA
Enzyme Induction
Proteins
Glutathione
Antioxidants
Western Blotting

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Induction of thioltransferase and thioredoxin/thioredoxin reductase systems in cultured porcine lenses under oxidative stress. / Moon, Sungchur; Fernando, M Rohan; Lou, Marjorie F.

In: Investigative Ophthalmology and Visual Science, Vol. 46, No. 10, 01.10.2005, p. 3783-3789.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. In view of the important antioxidant roles of thioltransferase (TTase), thioredoxin (Trx), and thioredoxin reductase (TR) in the lens, the present study was conducted to investigate the induction of these cytosolic enzymes in response to H2O2 stress in cultured lenses. METHODS. Porcine lenses were cultured, exposed to H2O2 for various lengths of time between 0 and 24 hours, and photographed to detect morphologic changes. The lenses were then harvested; dissected into epithelial layer, cortex, and nucleus; and homogenized for the determination of the glutathione (GSH) level. Pooled epithelial layers were used to examine TTase, Trx, and TR protein or mRNA levels. RESULTS. Treatment of lenses with H 2O2 caused distinct morphologic changes. Lower concentrations of H2O2 (0.2 mM) caused the lens to be hazy after 6 hours and to worsen progressively between 12 and 24 hours. Higher levels of H2O2 (0.5 mM) induced similar morphologic changes, but sooner (within 1 hour) and more severe. Both H2O 2-treated groups showed a dramatic and gradual GSH depletion during the 24-hour incubation, but the GSH level at 50{\%} or above appeared to be essential in maintaining lens clarity. However, TTase, Trx, and TR activities, protein expressions, and mRNA transcriptions in the epithelial layers of these lenses were increased, but each enzyme had a distinct pattern. Under mild H 2O2 stress, a slow and transient activation of TTase, Trx, and TR was observed. However, under stronger H2O2 stress, all three enzymes showed a very rapid increase and then a steady decline in activity. Western blot and RT-PCR analyses revealed that this increase in activity in all three enzymes was due to the induction of protein and mRNA expression. In the control group (no oxidative stress) all three enzyme activities and their respective expressions remained constant throughout the experimental period. CONCLUSIONS. The data show that TTase, Trx, and TR activity and expression are induced in lens cells under oxidative stress, probably to protect and maintain the health of the lens.",
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N2 - PURPOSE. In view of the important antioxidant roles of thioltransferase (TTase), thioredoxin (Trx), and thioredoxin reductase (TR) in the lens, the present study was conducted to investigate the induction of these cytosolic enzymes in response to H2O2 stress in cultured lenses. METHODS. Porcine lenses were cultured, exposed to H2O2 for various lengths of time between 0 and 24 hours, and photographed to detect morphologic changes. The lenses were then harvested; dissected into epithelial layer, cortex, and nucleus; and homogenized for the determination of the glutathione (GSH) level. Pooled epithelial layers were used to examine TTase, Trx, and TR protein or mRNA levels. RESULTS. Treatment of lenses with H 2O2 caused distinct morphologic changes. Lower concentrations of H2O2 (0.2 mM) caused the lens to be hazy after 6 hours and to worsen progressively between 12 and 24 hours. Higher levels of H2O2 (0.5 mM) induced similar morphologic changes, but sooner (within 1 hour) and more severe. Both H2O 2-treated groups showed a dramatic and gradual GSH depletion during the 24-hour incubation, but the GSH level at 50% or above appeared to be essential in maintaining lens clarity. However, TTase, Trx, and TR activities, protein expressions, and mRNA transcriptions in the epithelial layers of these lenses were increased, but each enzyme had a distinct pattern. Under mild H 2O2 stress, a slow and transient activation of TTase, Trx, and TR was observed. However, under stronger H2O2 stress, all three enzymes showed a very rapid increase and then a steady decline in activity. Western blot and RT-PCR analyses revealed that this increase in activity in all three enzymes was due to the induction of protein and mRNA expression. In the control group (no oxidative stress) all three enzyme activities and their respective expressions remained constant throughout the experimental period. CONCLUSIONS. The data show that TTase, Trx, and TR activity and expression are induced in lens cells under oxidative stress, probably to protect and maintain the health of the lens.

AB - PURPOSE. In view of the important antioxidant roles of thioltransferase (TTase), thioredoxin (Trx), and thioredoxin reductase (TR) in the lens, the present study was conducted to investigate the induction of these cytosolic enzymes in response to H2O2 stress in cultured lenses. METHODS. Porcine lenses were cultured, exposed to H2O2 for various lengths of time between 0 and 24 hours, and photographed to detect morphologic changes. The lenses were then harvested; dissected into epithelial layer, cortex, and nucleus; and homogenized for the determination of the glutathione (GSH) level. Pooled epithelial layers were used to examine TTase, Trx, and TR protein or mRNA levels. RESULTS. Treatment of lenses with H 2O2 caused distinct morphologic changes. Lower concentrations of H2O2 (0.2 mM) caused the lens to be hazy after 6 hours and to worsen progressively between 12 and 24 hours. Higher levels of H2O2 (0.5 mM) induced similar morphologic changes, but sooner (within 1 hour) and more severe. Both H2O 2-treated groups showed a dramatic and gradual GSH depletion during the 24-hour incubation, but the GSH level at 50% or above appeared to be essential in maintaining lens clarity. However, TTase, Trx, and TR activities, protein expressions, and mRNA transcriptions in the epithelial layers of these lenses were increased, but each enzyme had a distinct pattern. Under mild H 2O2 stress, a slow and transient activation of TTase, Trx, and TR was observed. However, under stronger H2O2 stress, all three enzymes showed a very rapid increase and then a steady decline in activity. Western blot and RT-PCR analyses revealed that this increase in activity in all three enzymes was due to the induction of protein and mRNA expression. In the control group (no oxidative stress) all three enzyme activities and their respective expressions remained constant throughout the experimental period. CONCLUSIONS. The data show that TTase, Trx, and TR activity and expression are induced in lens cells under oxidative stress, probably to protect and maintain the health of the lens.

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