Roberto Iorio

Extremely low frequency electromagnetic fields (ELF‐EMFs) induce in vitro angiogenesis process in human endothelial cells

Effects of extremely low frequency (ELF) electromagnetic fields (EMFs) on activation of angiogenesis were analysed using cultured umbilical human vein endothelial cells (HUVECs). The cultures were exposed to a sinusoidal EMF to intensity of 1 mT, 50 Hz for up to 12 h. EMFs increased the degree of endothelial cell proliferation and tubule formation, coupled by an acceleration in the process of wound healing. Since this process is physiologically accompanied by a large modification in the structural organization of actin and focal adhesions, we analyzed the rearrangement of some cytoskeleton elements demonstrating a major reorganization of the fibres and of the focal adhesion complexes after EMF exposure. Finally, Western blot analysis revealed a significant increase in phosphorylation as well as the overall expression of VEGF receptor 2 (KDR/Flk-1) suggesting that EMFs may modulate in vitro some endothelial functions correlated to angiogenesis through signal transduction pathways dependent on VEGF.

Involvement of mitochondrial activity in mediating ELF-EMF stimulatory effect on human sperm motility

It has recently been reported that the exposure of human spermatozoa to an extremely low frequency (ELF) electromagnetic field (EMF) with a square waveform of 5 mT amplitude and frequency of 50 Hz improves sperm motility. The functional relationship between the energy metabolism and the enhancement of human sperm motility induced by ELF-EMF was investigated. Sperm exposure to ELF-EMF resulted in a progressive and significant increase of mitochondrial membrane potential and levels of ATP, ADP and NAD(+) that was associated with a progressive and significant increase in the sperm kinematic parameters. No significant effects were detected on other parameters such as ATP/ADP ratio and energy charge. When carbamoyl cyanide m-chlorophenylhydrazone (CICCP) was applied to inhibit the oxidative phosphorylation in the mitochondria, the values of energy parameters and motility in the sperm incubated in the presence of glucose and exposed to ELF-EMF did not change, thus indicating that the glycolysis was not involved in mediating ELF-EMF stimulatory effect on motility. By contrast, when pyruvate and lactate were provided instead of glucose, the energy status and motility increased significantly in ELF-EMF-treated sperm. Under these culture conditions, the inhibition of glycolitic metabolism by 2-deoxy-D-glucose (DOG) again resulted in increased values of energy and kinematic parameters, indicating that gluconeogenesis was not involved in producing glucose for use in glycolysis. We concluded that the key role in mediating the stimulatory effects exerted by ELF-EMF on human sperm motility is played by mitochondrial oxidative phosphorylation rather than glycolysis.

Ovarian Toxicity: From Environmental Exposure to Chemotherapy

Unlike men, who have continuous spermatogenesis throughout most of their lifetime, women are born with a fixed supply of follicles, and this number progressively declines with age until the menopause. Beside age, the speed of follicle depletion can be regulated by genetic, hormonal and environmental influences. In the course of their lives, women are exposed to multiple chemicals and radiation sources that can increase the chance of developing permanent infertility and premature ovarian failure (POF). A wealth of experimental data indicate that iatrogenic (chemotherapy, radiotherapy) and xenobiotic agents (e.g., chemicals, pharmaceuticals) are potent ovotoxicants capable of accelerating ovarian reserve depletion. In the present review we reported the negative effects exerted on mammalian ovary by some widely diffused environmental chemicals, as polycyclic aromatic hydrocarbons (PAHs) and dithiocarbamate mancozeb, and by 1-3 butadiene and 4-vinylcycloexene, two occupational chemicals known to be capable of inducing ovarian cancer and infertility. Furthermore, attention has been devoted to the consequences of chemo- and radiotherapy on the ovary, both known to affect reproductive lifespan. Our increasing understanding of metabolic alterations induced by these agents is fundamental to individuate new therapeutic strategies aimed to prevent ovarian dysfunction in fertile women.

Inhibition of Angiogenesis Mediated by Extremely Low- Frequency Magnetic Fields (ELF-MFs)

The formation of new blood vessels is an essential therapeutic target in many diseases such as cancer, ischemic diseases, and chronic inflammation. In this regard, extremely low-frequency (ELF) electromagnetic fields (EMFs) seem able to inhibit vessel growth when used in a specific window of amplitude. To investigate the mechanism of anti-angiogenic action of ELF-EMFs we tested the effect of a sinusoidal magnetic field (MF) of 2 mT intensity and frequency of 50 Hz on endothelial cell models HUVEC and MS-1 measuring cell status and proliferation, motility and tubule formation ability. MS-1 cells when injected in mice determined a rapid tumor-like growth that was significantly reduced in mice inoculated with MF-exposed cells. In particular, histological analysis of tumors derived from mice inoculated with MF-exposed MS-1 cells indicated a reduction of hemangioma size, of blood-filled spaces, and in hemorrhage. In parallel, in vitro proliferation of MS-1 treated with MF was significantly inhibited. We also found that the MF-exposure down-regulated the process of proliferation, migration and formation of tubule-like structures in HUVECs. Using western blotting and immunofluorescence analysis, we collected data about the possible influence of MF on the signalling pathway activated by the vascular endothelial growth factor (VEGF). In particular, MF exposure significantly reduced the expression and activation levels of VEGFR2, suggesting a direct or indirect influence of MF on VEGF receptors placed on cellular membrane. In conclusion MF reduced, in vitro and in vivo, the ability of endothelial cells to form new vessels, most probably affecting VEGF signal transduction pathway that was less responsive to activation. These findings could not only explain the mechanism of anti-angiogenic action exerted by MFs, but also promote the possible development of new therapeutic applications for treatment of those diseases where excessive angiogenesis is involved.

Mancozeb affects mitochondrial activity, redox status and ATP production in mouse granulosa cells.

BACKGROUND Mancozeb (MZ) is a fungicide that belongs to the subclass of metal (Mn/Zn) ethylene-bis-dithiocarbamate pesticides. In mouse and human granulosa cells (GCs) exposed to MZ (0.01 μg/ml), morphological modifications and significant alterations of p53 expression level in comparison with control GCs were recorded. OBJECTIVES To investigate if MZ (0.01 μg/ml) induces oxidative stress and alters energy metabolism in exposed mouse GCs. RESULTS Following fungicide exposure, GCs showed low p53 content, a depolarized mitochondrial membrane potential (ΔΨm), as well as low ATP and reduced glutathione (GSH) levels associated with increased reactive oxygen species (ROS) generation. No remarkable differences on other parameters such as ATP/ADP ratio, energy charge, as well as induction of apoptosis and DNA damage were found. The activation of AKT and PDK1 kinases in MZ-treated cells was observed. Inhibition of ROS generation by the antioxidant N-acetylcysteine (NAC) restored a normal expression level of p53. CONCLUSIONS Our results demonstrate that the low dose of MZ here used induces a mild oxidative stress in GCs, and provides evidence for the possible involvement of AKT/PKB signaling pathway in triggering adaptive and survival response.

ELF-MF transiently increases skeletal myoblast migration: Possible role of calpain system

Abstract Purpose: Cell migration is crucial for myogenesis since it is required for the alignment and fusion of myoblast. Ca2+ signals are involved in regulating myoblast migration and an extremely low frequency (ELF) magnetic field (MF) increases intracellular calcium levels in C2C12 myoblast. This study was aimed at investigating whether ELF-MF could affect myoblast migration. As calpains contribute to the regulation of myoblast motility, the effect of ELF-MF on μ- and m-calpain was also investigated. Materials and methods: The effect of ELF-MF (1 mT; 50 Hz) on C2C12 cell motility was observed by wound-healing assay. Protein expression of calpains, calpastatin, myristoylated alanine-rich C-kinase substrate (MARCKS) and vinculin were examined by Western blot analysis. Casein zymography and immunofluorescence analysis were carried out to evaluate, respectively, activity levels of calpains and intracellular distribution of calpains, calpastatin and actin. Results: Exposure to ELF-MF resulted in a transient but significant increase of myoblast migration. This stimulatory effect was associated with a marked increase of μ- and m-calpain activity followed by the concomitant variation in their subcellular localization. No significant changes in intracellular distribution and protein levels of calpastatin were detected. Finally, a significant decrease of MARCKS expression and modifications of actin dynamics were reported. Conclusions: This study clearly outlines an involvement of calpains in ELF-MF-mediated myoblast migration.

Apoptotic effects of bovine apo‐lactoferrin on HeLa tumor cells

Lactoferrin (Lf), a cationic iron‐binding glycoprotein of 80 kDa present in body secretions, is known as a compound with marked antimicrobial activity. In the present study, the apoptotic effect of iron‐free bovine lactoferrin (apo‐bLf) on human epithelial cancer (HeLa) cells was examined in association with reactive oxygen species and glutathione (GSH) levels. Apoptotic effect of iron‐free bovine lactoferrin inhibited the growth of HeLa cells after 48 hours of treatment while the diferric‐bLf was ineffective in the concentration range tested (from 1 to 12.5 μM). Western blot analysis showed that key apoptotic regulators including Bax, Bcl‐2, Sirt1, Mcl‐1, and PARP‐1 were modulated by 1.25 μM of apo‐bLf. In the same cell line, apo‐bLf induced apoptosis together with poly (ADP‐ribose) polymerase cleavage, caspase activation, and a significant drop of NAD+. In addition, apo‐bLf–treated HeLa cells showed a marked increase of reactive oxygen species level and a significant GSH depletion. On the whole, apo‐bLf triggered apoptosis of HeLa cells upon oxygen radicals burst and GSH decrease.

VEGFR2 Expression Is Differently Modulated by Parity and Nulliparity in Mouse Ovary

Parity and nulliparity exert opposite effects on womens health, as parity is considered a protective factor for several reproductive diseases. This study is aimed at determining if ovarian VEGF and VEGFR2 expression are differently modulated in the ovaries of parous and nulliparous mice. To this end primiparous and nulliparous fertile mice were sacrificed at postovulatory stage. Whole ovaries, corpus luteum, and residual stromal tissues were analyzed to assess VEGF/VEGFR2 expression levels. Ovarian mRNA amounts of Vegfa (120 and 164) and Vegfr2 were comparable between primiparous and nulliparous mice; both isoforms and receptor were accumulated mainly in corpus luteum tissues. VEGF 120 and 164 protein accumulation and distribution mirrored that of mRNA. Conversely, VEGFR2 protein content was significantly higher in ovaries of nulliparous mice and was more efficiently phosphorylated in ovaries of primiparous mice. In both groups, VEGFR2 was preferentially expressed in corpus luteum, while its phosphorylated form was equally distributed in two somatic compartments. We suggest that parity influences VEGFR2/phospho-VEGFR2 expression and tissue distribution. This difference could be part of a more complex mechanism that at least in mice is activated after the first pregnancy and likely aims to preserve female health.