Emodin and [6]-gingerol lessen hypoxia-induced embryotoxicities in cultured mouse whole embryos via upregulation of hypoxia-inducible factor 1α and intracellular superoxide dismutases
Introduction
Although the avascular embryo is physiologically hypoxic (2–5% O2) before organogenesis [1], the developing embryo undergoes both aerobic and anaerobic metabolic pathways during organogenesis and the oxygen demand increases with gradual fetal growth [2]. Cultured mouse embryos require the increasing levels of oxygen such as 5% O2 on embryonic day (ED) 8.5, 20% O2 on ED 9.5, 40% O2 on ED 10.5, and 95% O2 after ED 11.5 [3]. However, maternal factors including anemia, pulmonary disease, diabetes, drug-induced uterine vessel constriction (epinephrine, nicotine, or cocaine), and ethanol intake elicit fetal hypoxia that can cause low birth weight, growth restriction, infant mortality, and cardiovascular disease [4], [5], [6]. Therefore, excess hypoxia leads to developmental abnormalities and postnatal deficits [7].
It has been shown that hypoxia and hypoxia-inducible factor-1α (HIF-1α) are involved in the same signaling pathway and that both play essential roles in particular developmental processes [8]. HIF-1α can regulate expression of genes, many of which pertain to the enhancement of processes such as glucose utilization, erythropoiesis, angiogenesis, and cell survival and/or growth [9]. Therefore, HIF-1α is considered to be essential for cellular and developmental aspects of O2 homeostasis, the formation of key physiological systems during embryonic development, and their subsequent utilization during postnatal life [6].
Normally, oxygen plays a major role as an oxidant in the form of superoxides (O2−), hydroxyl radicals (OH−), and hydrogen peroxide (H2O2), collectively known as reactive oxygen species (ROS). Oxidative stress, an imbalance between ROS production and antioxidant defense mechanisms of a cell or tissue, leads to significant lipid peroxidation, RNA and DNA mutation, oxidation of proteins, and inactivation of many enzymes [7], [10]. Organisms have a complex antioxidant system composed of non-enzymatic and enzymatic mechanisms to scavenge ROS during hypoxia. Members of the superoxide dismutase (SOD) family, a ubiquitously distributed group of enzymes that efficiently catalyze the dismutation of O2−, are involved in a representative antioxidant defense system [11]. Cytoplasmic Cu/Zn SOD (SOD1) is located in multiple intracellular compartments including the cytosol, nucleus, lysosomes, and mitochondrial intermembrane space [12], [13], while the manganese SOD (SOD2) is only located in mitochondria [14].
Spices and herbs are recognized as sources of natural antioxidants that can provide protection from oxidative stress and thus play an important role in chemoprevention of diseases in which ROS have key roles in the etiology and pathophysiology [15]. Emodin is a naturally occurring anthraquinone found in the roots and bark of numerous plants of the genus Rhamnus which has anti-mutagenic, anti-cancer, anti-diuretic, vasorelaxant, and immunosuppressive activities [16], [17]. [6]-gingerol is a major gingerol which has antioxidant, anti-inflammatory, and anti-carcinogenic properties [18], [19], [20], [21]. Additionally, mimetics of manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a novel, stable and cell-permeable SOD, possess both SOD and catalase activities [22].
Hypoxic conditions due to exposure to various teratogens have increased among pregnant women and cause subsequent birth defects [7]. The purpose of the current study was to determine whether emodin and [6]-gingerol affects hypoxia-induced anomalies during embryonic organogenesis. To do this, we cultured embryonic day 8.5 mouse embryos under hypoxic conditions (5% O2) for 2 days with or without emodin, [6]-gingerol, and the SOD mimetic MnTBAP. We then investigated the developmental changes and expression patterns of hypoxia-inducible factor 1α (HIF-1α) and intracellular SODs in the embryos.
Section snippets
Chemicals and animals
Emodin (Sigma, St. Louis, MO, USA) and [6]-gingerol (Wako Chemicals, Osaka, USA) were diluted with dimethylsulfoxide (Amresco, St. Louis, USA) to a concentration of less than 0.001%. MnTBAP was purchased from Calbiochem (Darmstadt, Germany) and was diluted with phosphate-buffered saline. Male and female ICR mice (8–10 weeks old) were purchased from a commercial breeder, Biogenomics Co. (Seoul, South Korea). One male and three female mice were housed in a cage for mating. The environmental
Ontogenetic changes in the hypoxic mouse embryos cultured under presence or absence of exogenous antioxidants
As shown in Fig. 1 and Table 1, abnormalities and a significant decrease of the total morphological scores were observed in embryos exposed to hypoxic condition during a critical organogenic stage compared to normoxic embryos (P < 0.05). Hypoxic embryos exhibited severe developmental abnormalities in most of the organs examined. In particular, hypoxic embryos had significantly lower morphological scores for their yolk sac diameter and circulation, allantois, flexion, heart, hindbrain, midbrain,
Discussion
During pregnancy, maternal metabolic disturbances which lead to placental hypoxia have potentially detrimental effects on embryonic development [6], [28]. Although low oxygen tension in early stages of normal fetus development is necessary for normal vascularization in the uterus, chronic reductions in oxygen tension impair growth rates in the developing embryo [6]. Exposure to continuous hypoxia during organogenesis results in growth retardation of the fetus, decreases in anterior neuropore
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0029709).
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