Oxidative Stress and NADPH Oxidase: Connecting Electromagnetic Fields, Cation Channels and Biological Effects.

Electromagnetic fields (EMFs) disrupt the electrochemical balance of biological membranes, thereby causing abnormal cation movement and deterioration of the function of membrane voltage-gated ion channels. These can trigger an increase of oxidative stress (OS) and the impairment of all cellular functions, including DNA damage and subsequent carcinogenesis. In this review we focus on the main mechanisms of OS generation by EMF-sensitized NADPH oxidase (NOX), the involved OS biochemistry, and the associated key biological effects.

Mobile phone electromagnetic radiation affects Amyloid Precursor Protein and α-synuclein metabolism in SH-SY5Y cells.

In this study, the effects of low-level, GSM emitted ElectroMagnetic Field (EMF) on Amyloid Precursor Protein (APP) and alpha-synuclein (α-syn) in human neuroblastoma cells was investigated. Our data indicated alterations on APP processing and cellular topology, following EMF exposure (ℇ = 10.51 V/m, SAR = 0.23 W/kg, exposure time: 3 times, for 10 min, for 2 days). Furthermore, changes in monomeric α-syn accumulation and multimerization, as well as induction of oxidative stress and cell death, were documented. The results presented here require further investigation to determine potential links of EMF with the molecular pathogenic mechanisms in Alzheimer's and Parkinson's Diseases.

Applying Broadband Dielectric Spectroscopy (BDS) for the Biophysical Characterization of Mammalian Tissues under a Variety of Cellular Stresses.

The dielectric properties of biological tissues can contribute non-invasively to a better characterization and understanding of the structural properties and physiology of living organisms. The question we asked, is whether these induced changes are effected by an endogenous or exogenous cellular stress, and can they be detected non-invasively in the form of a dielectric response, e.g., an AC conductivity switch in the broadband frequency spectrum. This study constitutes the first methodological approach for the detection of environmental stress-induced damage in mammalian tissues by the means of broadband dielectric spectroscopy (BDS) at the frequencies of 1-106 Hz. Firstly, we used non-ionizing (NIR) and ionizing radiation (IR) as a typical environmental stress. Specifically, rats were exposed to either digital enhanced cordless telecommunication (DECT) radio frequency electromagnetic radiation or to γ-radiation, respectively. The other type of stress, characterized usually by high genomic instability, was the pathophysiological state of human cancer (lung and prostate). Analyzing the results of isothermal dielectric measurements provided information on the tissues' water fraction. In most cases, our methodology proved sufficient in detecting structural changes, especially in the case of IR and malignancy. Useful specific dielectric response patterns are detected and correlated with each type of stress. Our results point towards the development of a dielectric-based methodology for better understanding and, in a relatively invasive way, the biological and structural changes effected by radiation and developing lung or prostate cancer often associated with genomic instability.

Apoptotic cell death during Drosophila oogenesis is differentially increased by electromagnetic radiation depending on modulation, intensity and duration of exposure.

Present generations are being repeatedly exposed to different types and doses of non-ionizing radiation (NIR) from wireless technologies (FM radio, TETRA and TV stations, GSM and UMTS phones/base stations, Wi-Fi networks, DECT phones). Although there is controversy on the published data regarding the non-thermal effects of NIR, studies have convincingly demonstrated bioeffects. Their results indicate that modulation, intensity, exposure duration and model system are important factors determining the biological response to irradiation. Attempting to address the dependence of NIR bioeffectiveness on these factors, apoptosis in the model biological system Drosophila melanogaster was studied under different exposure protocols. A signal generator was used operating alternatively under Continuous Wave (CW) or Frequency Modulation (FM) emission modes, at three power output values (10 dB, 0, -10 dB), under four carrier frequencies (100, 395, 682, 900 MHz). Newly emerged flies were exposed either acutely (6 min or 60 min on the 6th day), or repeatedly (6 min or 60 min daily for the first 6 days of their life). All exposure protocols resulted in an increase of apoptotic cell death (ACD) observed in egg chambers, even at very low electric field strengths. FM waves seem to have a stronger effect in ACD than continuous waves. Regarding intensity and temporal exposure pattern, EMF-biological tissue interaction is not linear in response. Intensity threshold for the induction of biological effects depends on frequency, modulation and temporal exposure pattern with unknown so far mechanisms. Given this complexity, translating such experimental data into possible human exposure guidelines is yet arbitrary.

Reactive oxygen species elevation and recovery in Drosophila bodies and ovaries following short-term and long-term exposure to DECT base EMF.

The objective of this study was to approach the basic mechanism(s) underlying reported ovarian apoptotic cell death and fecundity decrease induced by nonionizing radiation (NIR) in Drosophila melanogaster. ROS (Reactive Oxygen Species) levels were measured in the bodies and the ovaries of (sexually mature) 4-day-old flies, following exposure for 0.5, 1, 6, 24 and 96 h to a wireless DECT (Digital Enhanced Cordless Telephone) base radiation (1.88-1.90 GHz). Electrical field intensity was 2.7 V/m, measured within the fly vials and calculated SAR (Specific Absorption Rate) value = 0.009 W/Kg. Male and female bodies showed twofold increase in ROS levels (p < 0.001) after 6 h of exposure, slightly increasing with more irradiation (24 and 96 h). Ovaries of exposed females had a quick response in ROS increase after 0.5 h (1.5-fold, p < 0.001), reaching 2.5-fold after 1 h with no elevation thereafter at 6, 24 and 96 h. ROS levels returned to normal, in the male and the female bodies 24 h after 6 h of exposure of the flies (p < 0.05) and in the ovaries 4 h after 1 h exposure of the females (p < 0.05). It is postulated that the pulsed (at 100 Hz rate and 0.08 ms duration) idle state of the DECT base radiation is capable of inducing free radical formation albeit the very low SAR, leading rapidly to accumulation of ROS in a level-saturation manner under continuous exposure, or in a recovery manner after interruption of radiation, possibly due to activation of the antioxidant machinery of the organism.

Drosophila oogenesis as a bio-marker responding to EMF sources.

The model biological organisms Drosophila melanogaster and Drosophila virilis have been utilized to assess effects on apoptotic cell death of follicles during oogenesis and reproductive capacity (fecundity) decline. A total of 280 different experiments were performed using newly emerged flies exposed for short time daily for 3-7 d to various EMF sources including: GSM 900/1800 MHz mobile phone, 1880-1900 MHz DECT wireless base, DECT wireless handset, mobile phone-DECT handset combination, 2.44 GHz wireless network (Wi-Fi), 2.44 GHz blue tooth, 92.8 MHz FM generator, 27.15 MHz baby monitor, 900 MHz CW RF generator and microwave oven's 2.44 GHz RF and magnetic field components. Mobile phone was used as a reference exposure system for evaluating factors considered very important in dosimetry extending our published work with D. melanogaster to the insect D. virilis. Distance from the emitting source, the exposure duration and the repeatability were examined. All EMF sources used created statistically significant effects regarding fecundity and cell death-apoptosis induction, even at very low intensity levels (0.3 V/m blue tooth radiation), well below ICNIRP's guidelines, suggesting that Drosophila oogenesis system is suitable to be used as a biomarker for exploring potential EMF bioactivity. Also, there is no linear cumulative effect when increasing the duration of exposure or using one EMF source after the other (i.e. mobile phone and DECT handset) at the specific conditions used. The role of the average versus the peak E-field values as measured by spectrum analyzers on the final effects is discussed.

Detrimental effects of proteasome inhibition activity in Drosophila melanogaster: implication of ER stress, autophagy, and apoptosis.

In eukaryotes, the ubiquitin-proteasome machinery regulates a number of fundamental cellular processes through accurate and tightly controlled protein degradation pathways. We have, herein, examined the effects of proteasome functional disruption in Dmp53 (+/+) (wild-type) and Dmp53 (-/-) Drosophila melanogaster fly strains through utilization of Bortezomib, a proteasome-specific inhibitor. We report that proteasome inhibition drastically shortens fly life-span and impairs climbing performance, while it also causes larval lethality and activates developmentally irregular cell death programs during oogenesis. Interestingly, Dmp53 gene seems to play a role in fly longevity and climbing ability. Moreover, Bortezomib proved to induce endoplasmic reticulum (ER) stress that was able to result in the engagement of unfolded protein response (UPR) signaling pathway, as respectively indicated by fly Xbp1 activation and Ref(2)P-containing protein aggregate formation. Larva salivary gland and adult brain both underwent strong ER stress in response to Bortezomib, thus underscoring the detrimental role of proteasome inhibition in larval development and brain function. We also propose that the observed upregulation of autophagy operates as a protective mechanism to "counterbalance" Bortezomib-induced systemic toxicity, which is tightly associated, besides ER stress, with activation of apoptosis, mainly mediated by functional Drice caspase and deregulated dAkt kinase. The reduced life-span of exposed to Bortezomib flies overexpressing Atg1_RNAi or Atg18_RNAi supports the protective nature of autophagy against proteasome inhibition-induced stress. Our data reveal the in vivo significance of proteasome functional integrity as a major defensive system against cellular toxicity likely occurring during critical biological processes and morphogenetic courses.

Transient and cumulative memory impairments induced by GSM 1.8 GHz cell phone signal in a mouse model.

This study was designed to investigate the transient and cumulative impairments in spatial and non-spatial memory of C57Bl/6J mice exposed to GSM 1.8 GHz signal for 90 min daily by a typical cellular (mobile) phone at a specific absorption rate value of 0.11 W/kg. Free-moving male mice 2 months old were irradiated in two experimental protocols, lasting for 66 and for 148 days respectively. Each protocol used three groups of animals (n = 8 each for exposed, sham exposed and controls) in combination with two behavioural paradigms, the object recognition task and the object location task sequentially applied at different time points. One-way analysis of variance revealed statistically significant impairments of both types of memory gradually accumulating, with more pronounced effects on the spatial memory. The impairments persisted even 2 weeks after interruption of the 8 weeks daily exposure, whereas the memory of mice as detected by both tasks showed a full recovery approximately 1 month later. Intermittent every other day exposure for 1 month had no effect on both types of memory. The data suggest that visual information processing mechanisms in hippocampus, perirhinal and entorhinal cortex are gradually malfunctioning upon long-term daily exposure, a phenotype that persists for at least 2 weeks after interruption of radiation, returning to normal memory performance levels 4 weeks later. It is postulated that cellular repair mechanisms are operating to eliminate the memory affecting molecules. The overall contribution of several possible mechanisms to the observed cumulative and transient impairments in spatial and non-spatial memory is discussed.

Brain proteome response following whole body exposure of mice to mobile phone or wireless DECT base radiation.

The objective of this study was to investigate the effects of two sources of electromagnetic fields (EMFs) on the proteome of cerebellum, hippocampus, and frontal lobe in Balb/c mice following long-term whole body irradiation. Three equally divided groups of animals (6 animals/group) were used; the first group was exposed to a typical mobile phone, at a SAR level range of 0.17-0.37 W/kg for 3 h daily for 8 months, the second group was exposed to a wireless DECT base (Digital Enhanced Cordless Telecommunications/Telephone) at a SAR level range of 0.012-0.028 W/kg for 8 h/day also for 8 months and the third group comprised the sham-exposed animals. Comparative proteomics analysis revealed that long-term irradiation from both EMF sources altered significantly (p < 0.05) the expression of 143 proteins in total (as low as 0.003 fold downregulation up to 114 fold overexpression). Several neural function related proteins (i.e., Glial Fibrillary Acidic Protein (GFAP), Alpha-synuclein, Glia Maturation Factor beta (GMF), and apolipoprotein E (apoE)), heat shock proteins, and cytoskeletal proteins (i.e., Neurofilaments and tropomodulin) are included in this list as well as proteins of the brain metabolism (i.e., Aspartate aminotransferase, Glutamate dehydrogenase) to nearly all brain regions studied. Western blot analysis on selected proteins confirmed the proteomics data. The observed protein expression changes may be related to brain plasticity alterations, indicative of oxidative stress in the nervous system or involved in apoptosis and might potentially explain human health hazards reported so far, such as headaches, sleep disturbance, fatigue, memory deficits, and brain tumor long-term induction under similar exposure conditions.

Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.

alpha-Synuclein is central in Parkinson's disease pathogenesis. Although initially alpha-synuclein was considered a purely intracellular protein, recent data suggest that it can be detected in the plasma and CSF of humans and in the culture media of neuronal cells. To address a role of secreted alpha-synuclein in neuronal homeostasis, we have generated wild-type alpha-synuclein and beta-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of alpha-synuclein are readily detected in the conditioned media (CM) of these cells at concentrations similar to those observed in human CSF. We have found that, in this model, alpha-synuclein is secreted by externalized vesicles in a calcium-dependent manner. Electron microscopy and liquid chromatography-mass spectrometry proteomic analysis demonstrate that these vesicles have the characteristic hallmarks of exosomes, secreted intraluminar vesicles of multivesicular bodies. Application of CM containing secreted alpha-synuclein causes cell death of recipient neuronal cells, which can be reversed after alpha-synuclein immunodepletion from the CM. High- and low-molecular-weight alpha-synuclein species, isolated from this CM, significantly decrease cell viability. Importantly, treatment of the CM with oligomer-interfering compounds before application rescues the recipient neuronal cells from the observed toxicity. Our results show for the first time that cell-produced alpha-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that alpha-synuclein secretion serves to amplify and propagate Parkinson's disease-related pathology.

Programmed cell death of the ovarian nurse cells during oogenesis of the ladybird beetle Adalia bipunctata (Coleoptera: Coccinellidae).

Programmed cell death (PCD) is an evolutionary conserved and genetically regulated form of cell death, in which the cell plays an active role in its own demise. It is widely recognized that PCD can be morphologically classified into three major types: type I, known as apoptosis, type II, called autophagy, and type III, specified as cytoplasmic cell death. So far, PCD has been morphologically analyzed in certain model insect species of the meroistic polytrophic ovary-type, but has never been examined before in insects carrying meroistic telotrophic ovaries. In the present study, we attempted to thoroughly describe the three different types (I, II and III) of PCD occurring during oogenesis in the meroistic telotrophic ovary of the Coleoptera species Adalia bipunctata, at different developmental ages of the adult female insects. We reveal that in the ladybird beetle A. bipunctata, the ovarian tropharia undergo age-dependent forms of apoptotic, autophagic and cytoplasmic (paraptotic-like) cell death, which seem to operate in a rather synergistic fashion, in accordance with previous observations in Diptera and Lepidoptera species. Furthermore, we herein demonstrate the occurrence of morphogenetically abnormal ovarioles in A. bipunctata female insects. These atretic ovarioles collapse and die through a PCD-mediated process that is characterized by the combined activation of all three types of PCD. Conclusively, the distinct cell death programs (I, II and III) specifically engaged during oogenesis of A. bipunctata provide strong evidence for the structural and functional conserved nature of PCD during insect evolution among meroistic telotrophic and meroistic polytrophic ovary-type insects.

Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis.

Ubiquitin/proteasome-mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing ß2 and ß6 conditional mutant proteasome subunits in a cell type-specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell-specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation. In vivo targeted overexpression of ß2 and ß6 conditional mutants, specifically in the nurse cell compartment, led to a notable up-regulation of sporadic apoptosis potency during early and mid-oogenesis 'checkpoints', thus reasonably justifying the observed reduction in eclosion efficiency. Furthermore, in response to the intracellular abundance of ß2 and ß6 conditional mutant forms, specifically in numerous tissues of third instar larval stage, the developmental course was arrested, and lethal phenotypes were obtained at this particular embryonic period, with the double transgenic heterozygote embryos being unable to further proceed to complete maturation to adult flies. Our data demonstrate that physiological proteasome function is required to ensure normal oogenesis and embryogenesis in D. melanogaster, since targeted and cell type-dependent proteasome inactivation initiates developmentally deregulated apoptotic and autophagic mechanisms.

17-Allylamino-17-demethoxygeldanamycin induces downregulation of critical Hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells.

BACKGROUND: 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a benzoquinone ansamycin antibiotic, specifically targets heat shock protein 90 (Hsp90) and interferes with its function as a molecular chaperone that maintains the structural and functional integrity of various protein clients involved in cellular signaling. In this study, we have investigated the effect of 17-AAG on the regulation of Hsp90-dependent signaling pathways directly implicated in cell cycle progression, survival and motility of human urinary bladder cancer cell lines. METHODS: We have used MTT-based assays, FACS analysis, Western blotting, semi-quantitative RT-PCR, immunocytochemistry and scratch-wound assay in RT4, RT112 and T24 human urinary bladder cancer cell lines. RESULTS: We have demonstrated that, upon 17-AAG treatment, bladder cancer cells are arrested in the G1 phase of the cell cycle and eventually undergo apoptotic cell death in a dose-dependent manner. Furthermore, 17-AAG administration was shown to induce a pronounced downregulation of multiple Hsp90 protein clients and other downstream effectors, such as IGF-IR, Akt, IKK-α, IKK-ß, FOXO1, ERK1/2 and c-Met, resulting in sequestration-mediated inactivation of NF-κB, reduced cell proliferation and decline of cell motility. CONCLUSIONS: In total, we have clearly evinced a dose-dependent and cell type-specific effect of 17-AAG on cell cycle progression, survival and motility of human bladder cancer cells, due to downregulation of multiple Hsp90 clients and subsequent disruption of signaling integrity.

Red blood cell aging markers during storage in citrate-phosphate-dextrose-saline-adenine-glucose-mannitol.

BACKGROUND: It has been suggested that red blood cell (RBC) senescence is accelerated under blood bank conditions, although neither protein profile of RBC aging nor the impact of additive solutions on it have been studied in detail. STUDY DESIGN AND METHODS: RBCs and vesicles derived from RBCs in both citrate-phosphate-dextrose (CPD)-saline-adenine-glucose-mannitol (SAGM) and citrate-phosphate-dextrose-adenine (CPDA) were evaluated for the expression of cell senescence markers (vesiculation, protein aggregation, degradation, activation, oxidation, and topology) through immunoblotting technique and immunofluorescence or immunoelectron microscopy study. RESULTS: A group of cellular stress proteins exhibited storage time- and storage medium-related changes in their membrane association and exocytosis. The extent, the rate, and the expression of protein oxidation, Fas oligomerization, caspase activation, and protein modifications in Band 3, hemoglobin, and immunoglobulin G were less conspicuous and/or exhibited significant time retardation under storage in CPD-SAGM, compared to the CPDA storage. There was evidence for the localization of activated caspases near to the membrane of both cells and vesicles. CONCLUSIONS: We provide circumstantial evidence for a lower protein oxidative damage in CPD-SAGM-stored RBCs compared to the CPDA-stored cells. The different expression patterns of the senescence markers in the RBCs seem to be accordingly related to the oxidative stress management of the cells. We suggest that the storage of RBCs in CPD-SAGM might be more alike the in vivo RBC aging process, compared to storage in CPDA, since it is characterized by a slower stimulation of the recognition signaling pathways that are already known to trigger the erythrophagocytosis of senescent RBCs.

Intracellular clusterin inhibits mitochondrial apoptosis by suppressing p53-activating stress signals and stabilizing the cytosolic Ku70-Bax protein complex.

PURPOSE: Secretory clusterin (sCLU)/apolipoprotein J is an extracellular chaperone that has been functionally implicated in DNA repair, cell cycle regulation, apoptotic cell death, and tumorigenesis. It exerts a prosurvival function against most therapeutic treatments for cancer and is currently an antisense target in clinical trials for tumor therapy. However, the molecular mechanisms underlying its function remained largely unknown. EXPERIMENTAL DESIGN: The molecular effects of small interfering RNA-mediated sCLU depletion in nonstressed human cancer cells were examined by focusing entirely on the endogenously expressed sCLU protein molecules and combining molecular, biochemical, and microscopic approaches. RESULTS: We report here that sCLU depletion in nonstressed human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells correlates to altered ratios of proapoptotic to antiapoptotic Bcl-2 protein family members, is amplified by p53, and is executed by mitochondrial dysfunction. sCLU depletion-related stress signals originate from several sites, because sCLU is an integral component of not only the secretory pathway but also the nucleocytosolic continuum and mitochondria. In the cytoplasm, sCLU depletion disrupts the Ku70-Bax complex and triggers Bax activation and relocation to mitochondria. We show that sCLU binds and thereby stabilizes the Ku70-Bax protein complex serving as a cytosol retention factor for Bax. CONCLUSIONS: We suggest that elevated sCLU levels may enhance tumorigenesis by interfering with Bax proapoptotic activities and contribute to one of the major characteristics of cancer cells, that is, resistance to apoptosis.

The effect of exposure duration on the biological activity of mobile telephony radiation.

In the present experiments we studied the effects of different durations of a single (continuous), daily exposure, ranging from 1 min up to 21 min, to the two established systems of digital mobile telephony radiation that are commonly used in Europe, viz. GSM 900 MHz (Global System for Mobile telecommunications) and DCS 1800 MHz (Digital Cellular System-referred to also as GSM 1800 MHz), on a well-tested biological model, the reproductive capacity of the insect Drosophila melanogaster. The insects were exposed to each type of radiation at an intensity of about 10 microW/cm(2), corresponding to a distance of 20 or 30 cm from the antenna of a DCS 1800 or a GSM 900 mobile phone handset, respectively. At these distances the bioactivity of mobile telephony radiation was found to be at a maximum due to the existence of a "window" of increased bioactivity around this value, as we have shown recently [1-4]. The results show that the reproductive capacity decreases almost linearly with increasing exposure duration to both GSM 900 and DCS 1800 radiation, suggesting that short-term exposures to these radiations have cumulative effects on living organisms. Additionally, our results show again that GSM 900 MHz radiation is slightly more bioactive than DCS 1800 MHz radiation, at the same exposure durations and under equal radiation intensities, as shown in our previous experiments [5].

Comparison of biological effects between continuous and intermittent exposure to GSM-900-MHz mobile phone radiation: Detection of apoptotic cell-death features.

In the present study we used a 6-min daily exposure of dipteran flies, Drosophila melanogaster, to GSM-900MHz (Global System for Mobile Telecommunications) mobile phone electromagnetic radiation (EMR), to compare the effects between the continuous and four different intermittent exposures of 6min total duration, and also to test whether intermittent exposure provides any cumulative effects on the insect's reproductive capacity as well as on the induction of apoptotic cell death. According to our previous experiments, a 6-min continuous exposure per day for 5 days to GSM-900MHz and DCS-1800MHz (Digital Cellular System) mobile phone radiation, brought about a large decrease in the insect's reproductive capacity, as defined by the number of F(1) pupae. This decrease was found to be non-thermal and correlated with an increased percentage of induced fragmented DNA in the egg chambers' cells at early- and mid-oogenesis. In the present experiments we show that intermittent exposure also decreases the reproductive capacity and alters the actin-cytoskeleton network of the egg chambers, another known aspect of cell death that was not investigated in previous experiments, and that the effect is also due to DNA fragmentation. Intermittent exposures with 10-min intervals between exposure sessions proved to be almost equally effective as continuous exposure of the same total duration, whereas longer intervals between the exposures seemed to allow the organism the time required to recover and partly overcome the above-mentioned effects of the GSM exposure.

Scientific panel on electromagnetic field health risks: consensus points, recommendations, and rationales.

In November, 2009, a scientific panel met in Seletun, Norway, for three days of intensive discussion on existing scientific evidence and public health implications of the unprecedented global exposures to artificial electromagnetic fields (EMF). EMF exposures (static to 300 GHz) result from the use of electric power and from wireless telecommunications technologies for voice and data transmission, energy, security, military and radar use in weather and transportation. The Scientific Panel recognizes that the body of evidence on EMF requires a new approach to protection of public health; the growth and development of the fetus, and of children; and argues for strong preventative actions. New, biologically-based public exposure standards are urgently needed to protect public health worldwide.

Exploitation of Drosophila Choriogenesis Process as a Model Cellular System for Assessment of Compound Toxicity: the Phloroglucinol Paradigm.

Phloroglucinol (1,3,5 tri-hydroxy-benzene) (PGL), a natural phenolic substance, is a peroxidase inhibitor and has anti-oxidant, anti-diabetic, anti-inflammatory, anti-thrombotic, radio-protective, spasmolytic and anti-cancer activities. PGL, as a medicine, is administered to patients to control the symptoms of irritable bowel syndrome and acute renal colic, in clinical trials. PGL, as a phenolic substance, can cause cytotoxic effects. Administration of PGL up to 300 mg/kg (bw) is well tolerated by animals, while in cell lines its toxicity is developed at concentrations above the dose of 10 µg/ml. Furthermore, it seems that tumor or immortalized cells are more susceptible to the toxic power of PGL, than normal cells. However, studies of its cytotoxic potency, at the cellular level, in complex, differentiated and meta-mitotic biological systems, are still missing. In the present work, we have investigated the toxic activity of PGL in somatic epithelial cells, constituting the follicular compartment of a developing egg-chamber (or, follicle), which directs the choriogenesis (i.e. chorion assembly) process, during late oogenesis of Drosophila melanogaster. Our results reveal that treatment of in vitro growing Drosophila follicles with PGL, at a concentration of 0.2 mM (or, 25.2 µg/ml), does not lead to follicle-cell toxicity, since the protein-synthesis program and developmental pattern of choriogenesis are normally completed. Likewise, the 1 mM dose of PGL was also characterized by lack of toxicity, since the chorionic proteins were physiologically synthesized and the chorion structure appeared unaffected, except for a short developmental delay, being observed. In contrast, concentrations of 10, 20 or 40 mM of PGL unveiled a dose-dependent, increasing, toxic effect, being initiated by interruption of protein synthesis and disassembly of cell-secretory machinery, and, next, followed by fragmentation of the granular endoplasmic reticulum (ER) into vesicles, and formation of autophagic vacuoles. Follicle cells enter into an apoptotic process, with autophagosomes and large vacuoles being formed in the cytoplasm, and nucleus showing protrusions, granular nucleolus and condensed chromatin. PGL, also, proved able to induce disruption of nuclear envelope, activation of nucleus autophagy (nucleophagy) and formation of a syncytium-like pattern being produced by fusion of plasma membranes of two or more individual follicle cells. Altogether, follicle cell-dependent choriogenesis in Drosophila has been herein presented as an excellent, powerful and reliable multi-cellular, differentiated, model biological (animal) system for drug-cytotoxicity assessment, with the versatile compound PGL serving as a characteristic paradigm. In conclusion, PGL is a substance that may act beneficially for a variety of pathological conditions and can be safely used for differentiated somatic -epithelial- cells at clinically low concentrations. At relatively high doses, it could potentially induce apoptotic and autophagic cell death, thus being likely exploited as a therapeutic agent against a number of pathologies, including human malignancies.

Human bladder cancer cells undergo cisplatin-induced apoptosis that is associated with p53-dependent and p53-independent responses.

Cisplatin is a first-line chemotherapeutic agent and a powerful component of standard treatment regimens for several human malignancies including bladder cancer. DNA-Pt adducts produced by cisplatin are mainly responsible for cellular toxicity and induction of apoptosis. Identification of the mechanisms that control sensitivity to cisplatin is central to improving its therapeutic index and to successfully encountering the acquired resistance frequently emerging during therapy. In the present study, using MTT-based assays, Western blotting and semi-quantitative RT-PCR, we examined the apoptosis-related cellular responses to cisplatin exposure in two human urinary bladder cancer cell lines characterized by different malignancy grade and p53 genetic status. Both RT4 (grade I; wild-type p53) and T24 (grade III; mutant p53) cell types proved to be vulnerable to cisplatin apoptotic activity, albeit in a grade-dependent and drug dose-specific manner, as demonstrated by the proteolytic processing profiles of Caspase-8, Caspase-9, Caspase-3, and the Caspase repertoire characteristic substrates PARP and Lamin A/C, as well. The differential resistance of RT4 and T24 cells to cisplatin-induced apoptosis was associated with an RT4-specific phosphorylation (Ser15; Ser392) pattern of p53, together with structural amputations of the Akt and XIAP anti-apoptotic regulators. Furthermore, cisplatin administration resulted in a Granzyme B-mediated proteolytic cleavage of Hsp90 molecular chaperone, exclusively occurring in RT4 cells. To generate functional networks, expression analysis of a number of genes, including Bik, Bim, Bcl-2, FAP-1, Fas, FasL, TRAIL, Puma, Caspase-10, ATP7A, ATP7B and MRP1, was performed, strongly supporting the role of p53-dependent and p53-independent transcriptional responses in cisplatin-induced apoptosis of bladder cancer cells.