Significance of glutathione depletion and oxidative stress in early embryogenesis in glucose-induced rat embryo culture.
Recent studies have demonstrated the protective effects of supplementing free oxygen radical scavenging enzymes against hyperglycemia-induced embryonic malformations. In this study, the glutathione (GSH)-dependent protection system in hyperglycemia-induced embryopathy was investigated. Rat embryos at the early head-fold stage (day 9.5) cultured in 66.7 mmol/l glucose for 48 h showed significant growth retardation and an increase in the frequency of malformations. The concentration of GSH and activity of the rate-limiting GSH-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), significantly decreased in embryos exposed to hyperglycemia compared with controls (7.9 +/- 0.6 vs. 12.5 +/- 0.9 nmol/mg protein, P < 0.01 and 13.3 +/- 1.9 vs. 22.6 +/- 1.1 microU/mg protein, P < 0.01, respectively). Decreased activity of gamma-GCS in embryos exposed to hyperglycemia was associated with decreased expression of gamma-GCS mRNA levels. However, the activities of superoxide dismutase and glutathione peroxidase did not significantly change in these embryos. Extracellular and intracellular free oxygen radical formations estimated by Lucigenin-dependent chemoluminescence and flow cytometric analysis using 2',7'-dichlorofluorescein diacetate increased in isolated embryonic cells taken from embryos cultured under hyperglycemia. Supplementation of 2 mmol/l GSH ester into the hyperglycemic culture nearly restored GSH concentration in these embryos (11.9 +/- 0.5 vs. 12.5 +/- 0.9 nmol/mg protein) and reduced the formation of free oxygen radical species leading to almost complete normalization of growth retardation and embryonic dysmorphogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Trocino
R A
RA
First Department of Internal Medicine, Nagasaki University School of Medicine, Japan.
Akazawa
S
S
Ishibashi
M
M
Matsumoto
K
K
Matsuo
H
H
Yamamoto
H
H
Goto
S
S
Urata
Y
Y
Kondo
T
T
Nagataki
S
S
D000818:Animals; D015152:Blotting, Northern; D004622:Embryo, Mammalian / Q000187:drug effects / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry; D016166:Free Radical Scavengers / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D005721:Glutamate-Cysteine Ligase / Q000096:biosynthesis / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology / Q000502:physiology*; D005979:Glutathione Peroxidase / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006943:Hyperglycemia; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D008297:Male; D018384:Oxidative Stress; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D013482:Superoxide Dismutase / Q000378:metabolism
D016166:Free Radical Scavengers; D012333:RNA, Messenger; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D005982:Glutathione Transferase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D005947:Glucose