Book Review

Abstract

To investigate the potential adverse effects of mobile phone radiation, we studied reactive oxygen species (ROS), DNA damage and apoptosis in mouse embryonic fibroblasts (NIH/3T3) after intermittent exposure (5?min on/10?min off, for various durations from 0.5 to 8?h) to an 1800-MHz GSM-talk mode electromagnetic radiation (EMR) at an average specific absorption rate of 2?W/kg. A 2′,7′-dichlorofluorescin diacetate fluorescence probe was used to detect intracellular ROS levels, immunofluorescence was used to detect γH2AX foci as a marker for DNA damage, and flow cytometry was used to measure apoptosis. Our results showed a significant increase in intracellular ROS levels after EMR exposure and it reached the highest level at an exposure time of 1?h (p?p?相似文献   

Carbon monoxide activates NF-kappaB via ROS generation and Akt pathways to protect against cell death of hepatocytes

Heme oxygenase overexpression or exogenous carbon monoxide (CO) protects against hepatocyte apoptosis and fulminant hepatitis. The prevention of hepatocyte apoptosis by CO has been shown to require activation of NF-kappaB. The purpose of these investigations was to determine the mechanism of CO-induced hepatocyte NF-kappaB activation and protection against apoptosis. Primary rat or mouse hepatocytes and Hep3B cells were utilized. CO exposure was performed at 250 parts per million. Main outcome measures included cell viability, reactive oxygen species (ROS) generation, and changes in the levels of the intracellular antioxidants glutathione and ascorbate. Western blotting was performed for phospho-Akt, total Akt, and IkappaBalpha. NF-kappaB activation was determined by electrophoretic mobility shift assay and luciferase reporter assays. We found that CO treatment of hepatocytes prevents spontaneous apoptosis and leads to an increase in ROS production in association with Akt phosphorylation and IkappaB degradation. CO did not increase ROS production in respiration-deficient (rho0) Hep3B cells. Both Akt phosphorylation and IkappaB degradation can be inhibited by the addition of antioxidants. Furthermore, CO-induced NF-kappaB activation is reversed by phosphatidylinositol 3-kinase (PI3-K) inhibitor (LY294002) or antioxidants. Additionally, prevention of spontaneous hepatocyte apoptosis by CO is reversed by PI3-K inhibition and antioxidants. In conclusion, these data implicate a survival pathway of CO-induced ROS, Akt phosphorylation, and NF-kappaB activation in cultured hepatocytes. This pathway may prove to be important in maintenance of hepatic function in both physiological and pathophysiological conditions.     

The Effects of Mobile Phone Radiofrequency Radiation on Cochlear Stria Marginal Cells in Sprague–Dawley Rats

To investigate the possible mechanisms for biological effects of 1,800 MHz mobile radiofrequency radiation (RFR), the radiation-specific absorption rate was applied at 2 and 4 W/kg, and the exposure mode was 5 min on and 10 min off (conversation mode). Exposure time was 24 h short-term exposure. Following exposure, to detect cell DNA damage, cell apoptosis, and reactive oxygen species (ROS) generation, the Comet assay test, flow cytometry, DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) staining, and a fluorescent probe were used, respectively. Our experiments revealed that mobile phone RFR did not cause DNA damage in marginal cells, and the rate of cell apoptosis did not increase (P > 0.05). However, the production of ROS in the 4 W/kg exposure group was greater than that in the control group (P < 0.05). In conclusion, these results suggest that mobile phone energy was insufficient to cause cell DNA damage and cell apoptosis following short-term exposure, but the cumulative effect of mobile phone radiation still requires further confirmation. Activation of the ROS system plays a significant role in the biological effects of RFR. Bioelectromagnetics. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.     

Increased dexamethasone-induced apoptosis of thymocytes from mice exposed to long-term extremely low frequency magnetic fields

To address the effect of extremely low frequency electromagnetic fields on programmed cell death we assessed both the spontaneous and dexamethasone (Dex)-induced apoptosis of thymocytes and spleen cells from mice submitted to a long-term continuous exposure of a 0.4–1.0 μT 60 Hz magnetic field or an 8–20 μT direct current (DC) magnetic field. Dex-induced apoptosis but not spontaneous apoptosis was substantially increased in thymocytes from 0.4 to 1.0 μT 60 Hz field-exposed animals. Spontaneous apoptosis and Dex-induced apoptosis of spleen cells were not affected by the 0.4–1.0 μT 60 Hz field exposure. In addition, spontaneous apoptosis and Dex-induced apoptosis of thymocytes and spleen cells from mice exposed to an 8–20 μT DC field were similar to the controls. These findings represent the first demonstration that thymocytes from mice exposed to a long-term 0.4–1.0 μT 60 Hz field may show abnormal response to Dex apoptotic stimuli. Bioelectromagnetics 19:131–135, 1998. © 1998 Wiley-Liss, Inc.     

Time-varying magnetic fields of 60 Hz at 7 mT induce DNA double-strand breaks and activate DNA damage checkpoints without apoptosis

The potential genotoxic effect of a time-varying magnetic field (MF) on human cells was investigated. Upon continuous exposure of human primary fibroblast and cervical cancer cells to a 60 Hz MF at 7 mT for 10-60 min, no significant change in cell viability was observed. However, deoxyribonucleic acid (DNA) double-strand breaks (DSBs) were detected, and the DNA damage checkpoint pathway was activated in these cells without programmed cell death (called apoptosis). The exposure of human cells to a 60 Hz MF did not induce intracellular reactive oxygen species (ROS) production, suggesting that the observed DNA DSBs are not directly caused by ROS. We also compared the position and time dependency of DNA DSBs with numerical simulation of MFs. The Lorentz force and eddy currents in these experiments were numerically calculated to investigate the influence of each factor on DNA DSBs. The DNA DSBs mainly occurred at the central region, where the MF was strongest, after a 30-min exposure. After 90 min, however, the amount of DNA DSBs increased rapidly in the outer regions, where the eddy current and Lorentz force were strong.     

Acute exposure to 930 MHz CW electromagnetic radiation in vitro affects reactive oxygen species level in rat lymphocytes treated by iron ions

The aim of this study was to test the hypothesis that the 930 MHz continuous wave (CW) electromagnetic field, which is the carrier of signals emitted by cellular phones, affects the reactive oxygen species (ROS) level in living cells. Rat lymphocytes were used in the experiments. A portion of the lymphocytes was treated with iron ions to induce oxidative processes. Exposures to electromagnetic radiation (power density 5 W/m2, theoretical calculated SAR = 1.5 W/kg) were performed within a GTEM cell. Intracellular ROS were measured by the fluorescent probe dichlorofluorescin diacetate (DCF-DA). The results show that acute (5 and 15 min) exposure does not affect the number of produced ROS. If, however, FeCl2 with final concentration 10 microg/ml was added to the lymphocyte suspensions to stimulate ROS production, after both durations of exposure, the magnitude of fluorescence (ROS level during the experiment) was significantly greater in the exposed lymphocytes. The character of the changes in the number of free radicals observed in our experiments was qualitatively compatible with the theoretical prediction from the model of electromagnetic radiation effect on radical pairs.     

Dieldrin-induced oxidative stress and neurochemical changes contribute to apoptopic cell death in dopaminergic cells.

We examined the acute toxicity of dieldrin, a possible environmental risk factor of Parkinson's disease, in a dopaminergic cell model, PC12 cells, to determine early cellular events underlying the pesticide-induced degenerative processes. EC(50) for 1 h dieldrin exposure was 143 microM for PC12 cells, whereas EC(50) for non-dopaminergic cells was 292-351 microM, indicating that dieldrin is more toxic to dopaminergic cells. Dieldrin also induced rapid, dose-dependent releases of dopamine and its metabolite, DOPAC, resulting in depletion of intracellular dopamine. Additionally, dieldrin exposure caused depolarization of mitochondrial membrane potential in a dose-dependent manner. Flow cytometric analysis showed generation of reactive oxygen species (ROS) within 5 min of dieldrin treatment, and significant increases in lipid peroxidation were also detected following 1 h exposure. ROS generation was remarkably inhibited in the presence of SOD. Dieldrin-induced apoptosis was significantly attenuated by both SOD and MnTBAP (SOD mimetic), suggesting that dieldrin-induced superoxide radicals serve as important signals in initiation of apoptosis. Furthermore, pretreatment with deprenyl (MAO-inhibitor) or alpha-methyl-L-p-tyrosine (TH-inhibitor) also suppressed dieldrin-induced ROS generation and DNA fragmentation. Taken together, these results suggest that rapid release of dopamine and generation of ROS are early cellular events that may account for dieldrin-induced apoptotic cell death in dopaminergic cells.     

Characterization of apoptosis signal transduction pathways in HL-5 cardiomyocytes exposed to ischemia/reperfusion oxidative stress model

During ischemia/reperfusion (I/R), cardiomyocytes are exposed to sudden lack of nutrients and successively to radical oxygen species (ROS). In the present study, we used the HL-5 cardiac atrial myocyte cell line exposed to serum/glucose depletion added or not in H(2)O(2) to mimic ROS during ischemia, then replaced in their standard culture medium to simulate reperfusion. We investigated the effects of serum/glucose depletion combined or not to ROS exposure on AKT and MAP kinases activation to address the role of each event with respect to apoptosis. We demonstrate that serum/glucose depletion per se did not induce apoptosis when compared to ROS exposure. In particular, ROS recruited p38MAPK and JNK pathways. SB202190 preventing p38MAPK activity, partially protected HL-5 from apoptosis while blocking JNK, thanks to JNKI, further enhanced apoptosis. Blocking phosphatidylinositol (PI) 3-kinase with LY294002 or ERKs with U0126 was without consequence on apoptosis. Finally, BCL-2 and BCL-X(L/S) expression levels were analyzed in cells exposed to 1 h ischemia followed by 12-h reperfusion in the presence or not of SB202190; BCL-2, but not BCL-X(L/S), expression was decreased in ROS treated cells but SB202190 failed to restore BCL-2 level. Our data suggest that p38MAPK activation primarily mediates ROS-induced apoptosis while concomitant JNK activation would represent a scavenger pathway for cells trying to escape apoptosis.     

L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress,Autophagy, and Apoptosis

Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.     

Acetylcholine inhibits long-term hypoxia-induced apoptosis by suppressing the oxidative stress-mediated MAPKs activation as well as regulation of Bcl-2, c-IAPs,and caspase-3 in mouse embryonic stem cells

This study examined the effect of acetylcholine (ACh) on the hypoxia-induced apoptosis of mouse embryonic stem (ES) cells. Hypoxia (60 h) decreased both the cell viability and level of [³H] thymidine incorporation, which were prevented by a pretreatment with ACh. However, the atropine (ACh receptor [AChR] inhibitor) treatment blocked the protective effect of ACh. Hypoxia (90 min) increased the intracellular level of reactive oxygen species (ROS). On the other hand, ACh inhibited the hypoxia-induced increase in ROS, which was blocked by an atropine treatment. Subsequently, the hypoxia-induced ROS increased the level of p38 mitogen activated protein kinase (MAPK) and Jun-N-terminal kinase (JNK) phosphorylation, which were inhibited by the ACh pretreatment. Moreover, hypoxic exposure (90 min) increased the level of nuclear factor-κB (NF-κB) phosphorylation, which was blocked by a pretreatment with SB 203580 (p38 MAPK inhibitor) or SP 600125 (JNK inhibitor). However, hypoxia (60 h) decreased the protein levels of Bcl-2 and c-IAPs (cellular inhibitor of apoptosis proteins) but increased the level of caspase-3 activation. All these effects were inhibited by a pretreatment with ACh. In conclusion, ACh prevented the hypoxia-induced apoptosis of mouse ES cells by inhibiting the ROS-mediated p38 MAPK and JNK activation as well as the regulation of Bcl-2, c-IAPs, and caspase-3. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of negative air ions on respiratory organs and blood

The effect of negatively charged ions on respiratory organs and blood of rats has been studied. It was shown that the inhaling of negative air ions (NAI) for 60 min with a concentration of NAI at the place of location of animals 320-350 000 ions/cm2 activated the secretion of goblet cells without damaging the mucosa of the trachea and changed the spectrum of proteins of bronchopulmonary lavage. It was also found that the spontaneous production of reactive oxygen species (ROS) by cells of nonfractionated blood after the exposure to NAI increased in both males and females; the intensity of ROS generation induced by opsonized zymosan increased only in females. Different sensitivity of the antioxidant enzymes superoxide dismutase and glutathione reductase of blood to NAI in females and males was revealed. These results enable one to consider the effect of NAI as priming and a weak activation of the respiratory organs through the direct action on the mucosa of the primary target organs of the respiratory tract and then on the blood.     

The involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein (MAP) kinase in TRAIL/Apo2L-induced apoptosis

To determine the apoptotic signaling pathway which tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induced, we investigated the contribution of reactive oxygen species (ROS), p38 mitogen-activated protein (MAP) kinase and caspases in human adenocarcinoma HeLa cells. Here we show that upon TRAIL/Apo2L exposure there was pronounced ROS accumulation and activation of p38 MAP kinase, and that activation of caspases and apoptosis followed. Pretreatment with antioxidants such as glutathione or estrogen attenuated TRAIL/Apo2L-induced apoptosis through a reduction of ROS generation and diminished p38 MAP kinase and caspase activation. The p38 MAP kinase inhibitor SB203580 prevented apoptosis through a blockage of caspase activation, although ROS generation was not attenuated. Furthermore, the pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethyl ketone fully prevented apoptosis, while neither ROS accumulation nor p38 MAP kinase activation were affected. Therefore, our results suggest that TRAIL/Apo2L-induced apoptosis is mediated by ROS-activated p38 MAP kinase followed by caspase activation in HeLa cells.     

Coenzyme Q10 reduces ethanol-induced apoptosis in corneal fibroblasts

Dilute ethanol (EtOH) is a widely used agent to remove the corneal epithelium during the modern refractive surgery. The application of EtOH may cause the underlying corneal fibroblasts to undergo apoptosis. This study was designed to investigate the protective effect and potential mechanism of the respiratory chain coenzyme Q(10) (CoQ(10)), an electron transporter of the mitochondrial respiratory chain and a ubiquitous free radical scavenger, against EtOH-induced apoptosis of corneal fibroblasts. Corneal fibroblasts were pretreated with CoQ(10) (10 μM) for 2 h, followed by exposure to different concentrations of EtOH (0.4, 2, 4, and 20%) for 20 s. After indicated incubation period (2-12 h), MTT assay was used to examine cell viability. Treated cells were further assessed by flow cytometry to identify apoptosis. Reactive oxygen species (ROS) and the change in mitochondrial membrane potential were assessed using dichlorodihydrofluorescein diacetate/2',7'-dichlorofluorescein (DCFH-DA/DCF) assays and flow-cytometric analysis of JC-1 staining, respectively. The activity and expression of caspases 2, 3, 8, and 9 were evaluated with a colorimetric assay and western blot analysis. We found that EtOH treatment significantly decreased the viability of corneal fibroblasts characterized by a higher percentage of apoptotic cells. CoQ(10) could antagonize the apoptosis inducing effect of EtOH. The inhibition of cell apoptosis by CoQ(10) was significant at 8 and 12 h after EtOH exposure. In EtOH-exposed corneal fibroblasts, CoQ(10) pretreatment significantly reduced mitochondrial depolarization and ROS production at 30, 60, 90, and 120 min and inhibited the activation and expression of caspases 2 and 3 at 2 h after EtOH exposure. In summary, pretreatment with CoQ(10) can inhibit mitochondrial depolarization, caspase activation, and cell apoptosis. These findings support the proposition that CoQ(10) plays an antiapoptotic role in corneal fibroblasts after ethanol exposure.     

Mobile phone usage and male infertility in Wistar rats

A significant decrease in protein kinase C and total sperm count along with increased apoptosis were observed in male Wistar rats exposed to mobile phone frequencies (2 h/day x 35 days at 0.9 W/kg specific absorption rate). The results suggest that a reduction in protein kinase activity may be related to overproduction of reactive oxygen species (ROS) under microwave field exposure. Decrease in sperm count and an increase in apoptosis may be causative factor due to mobile radiation exposure leading to infertility.     

Mitochondrial-derived free radicals mediate asbestos-induced alveolar epithelial cell apoptosis

Asbestos causes pulmonary toxicity by mechanisms that in part involve reactive oxygen species (ROS). However, the precise source of ROS is unclear. We showed that asbestos induces alveolar epithelial cell (AEC) apoptosis by a mitochondrial-regulated death pathway. To determine whether mitochondrial-derived ROS are necessary for causing asbestos-induced AEC apoptosis, we utilized A549-rho(omicron) cells that lack mitochondrial DNA and a functional electron transport. As expected, antimycin, which induces an oxidative stress by blocking mitochondrial electron transport at complex III, increased dichlorofluoroscein (DCF) fluorescence in A549 cells but not in A549-rho(omicron) cells. Compared with A549 cells, rho(omicron) cells have less asbestos-induced ROS production, as assessed by DCF fluorescence, and reductions in total glutathione levels as well as less caspase-9 activation and apoptosis, as assessed by TdT-mediated dUTP nick end labeling staining and DNA fragmentation. A mitochondrial anion channel inhibitor that prevents ROS release from the mitochondria to the cytoplasm also blocked asbestos-induced A549 cell caspase-9 activation and apoptosis. Finally, a role for nonmitochondrial-derived ROS with exposure to high levels of asbestos (50 microg/cm(2)) was suggested by our findings that an iron chelator (phytic acid or deferoxamine) or a free radical scavenger (sodium benzoate) provided additional protection against asbestos-induced caspase-9 activation and DNA fragmentation in rho(omicron) cells. We conclude that asbestos fibers affect mitochondrial DNA and functional electron transport, resulting in mitochondrial-derived ROS production that in turn mediates AEC apoptosis. Nonmitochondrial-associated ROS may also contribute to AEC apoptosis, particularly with high levels of asbestos exposure.     

Mycobacterium tuberculosis promotes apoptosis in human neutrophils by activating caspase-3 and altering expression of Bax/Bcl-xL via an oxygen-dependent pathway

In addition to direct bactericidal activities, such as phagocytosis and generation of reactive oxygen species (ROS), neutrophils can regulate the inflammatory response by undergoing apoptosis. We found that infection of human neutrophils with Mycobacterium tuberculosis (Mtb) induced rapid cell death displaying the characteristic features of apoptosis such as morphologic changes, phosphatidylserine exposure, and DNA fragmentation. Both a virulent (H37Rv) and an attenuated (H37Ra) strain of Mtb were equally effective in inducing apoptosis. Pretreatment of neutrophils with antioxidants or an inhibitor of NADPH oxidase markedly blocked Mtb-induced apoptosis but did not affect spontaneous apoptosis. Activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis, but it was markedly augmented and accelerated during Mtb-induced apoptosis. The Mtb-induced apoptosis was associated with a speedy and transient increase in expression of Bax protein, a proapoptotic member of the Bcl-2 family, and a more prominent reduction in expression of the antiapoptotic protein Bcl-x(L). Pretreatment with an inhibitor of NADPH oxidase distinctly suppressed the Mtb-stimulated activation of caspase-3 and alteration of Bax/Bcl-x(L) expression in neutrophils. These results indicate that infection with Mtb causes ROS-dependent alteration of Bax/Bcl-x(L) expression and activation of caspase-3, and thereby induces apoptosis in human neutrophils. Moreover, we found that phagocytosis of Mtb-induced apoptotic neutrophils markedly increased the production of proinflammatory cytokine TNF-alpha by human macrophages. Therefore, the ROS-dependent apoptosis in Mtb-stimulated neutrophils may represent an important host defense mechanism aimed at selective removal of infected cells at the inflamed site, which in turn aids the functional activities of local macrophages.     

Reactive oxygen species formation is not enhanced by exposure to UMTS 1950 MHz radiation and co‐exposure to ferrous ions in Jurkat cells

This study was designed to assess if radiofrequency (RF) radiation induces oxidative stress in cultured mammalian cells when given alone or in combination with ferrous ions (FeSO₄). For this purpose the production of reactive oxygen species (ROS) was measured by flow cytometry in human lymphoblastoid cells exposed to 1950 MHz signal used by the third generation wireless technology of the Universal Mobile Telecommunication System (UMTS) at Specific Absorption Rate of 0.5 and 2.0 W/kg. Short (5–60 min) or long (24 h) duration exposures were carried out in a waveguide system under strictly controlled conditions of both dosimetry and environment. Cell viability was also measured after 24 h RF exposure using the Resazurin and Neutral Red assays. Several co‐exposure protocols were applied to test if RF radiation is able to alter ROS formation induced by FeSO₄ (RF given before or concurrently to FeSO₄). The results obtained indicate that non‐thermal RF exposures do not increase spontaneous ROS formation in any of the experimental conditions investigated. Consistent with the lack of ROS production, no change in cell viability was observed in Jurkat cells exposed to RF radiation for 24 h. Similar results were obtained when co‐exposures were considered: combined exposures to RF radiation and FeSO₄ did not increase ROS formation induced by the chemical treatment alone. In contrast, in cultures treated with FeSO₄ as positive control, a dose‐dependent increase in ROS formation was recorded, validating the sensitivity of the method employed. Bioelectromagnetics 30:525–535, 2009. © 2009 Wiley‐Liss, Inc.     

Globular adiponectin, acting via AdipoR1/APPL1, protects H9c2 cells from hypoxia/reoxygenation-induced apoptosis

Cardiomyocyte apoptosis is an important remodeling event contributing to heart failure and adiponectin may mediate cardioprotective effects at least in part via attenuating apoptosis. Here we used hypoxia-reoxygenation (H/R) induced apoptosis in H9c2 cells to examine the effect of adiponectin and cellular mechanisms of action. We first used TUNEL labeling in combination with laser scanning cytometry to demonstrate that adiponectin prevented H/R-induced DNA fragmentation. The anti-apoptotic effect of adiponectin was also verified via attenuation of H/R-induced phosphatidylserine exposure using annexin V binding. H/R-induced apoptosis via the mitochondrial-mediated intrinsic pathway of apoptosis as assessed by cytochrome c release into cytosol and caspase-3 activation, both of which were attenuated by adiponectin. Mechanistically, we demonstrated that adiponectin enhanced anti-oxidative potential in these cells which led to attenuation of the increase in intracellular reactive oxygen species (ROS) caused by H/R. To further address the mechanism of adiponctins anti-apoptotic effects we used siRNA to efficiently knockdown adiponectin receptor (AdipoR1) expression and found that this attenuated the protective effects of adiponectin on ROS production and caspase 3 activity. Knockdown of APPL1, an important intracellular binding partner for AdipoR, also significantly reduced the ability of adiponectin to prevent H/R-induced ROS generation and caspase 3 activity. In summary, H/R-induced ROS generation and activation of the intrinsic apoptotic pathway was prevented by adiponectin via AdipoR1/APPL1 signaling and increased anti-oxidant potential.     

Epidermal growth factor rescues trophoblast apoptosis induced by reactive oxygen species

Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast. This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin (hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT) and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants. The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with 10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal pregnancy.