In vitro effects of 1-MHz ultrasound on the mitotic spindle.

The effects of ultrasound on the cytoskeleton, comprising microtubules, had been studied decades ago. Nonetheless, very little attention has been paid to the effects of ultrasound on the mitotic spindle, which is also formed by microtubules. In this study, we treated human fibroblasts and human cancer cells (HeLa and MCF-7) with 1-MHz ultrasound at low intensities (70, 140, and 300 mW/cm2 ). In all cell lines, 5 min after the end of sonication, we found an intensity-dependent increase of mitotic abnormalities (including multipolar spindles). Two hours after sonication, these abnormalities were present, but at much lower frequencies. Twenty-four hours after sonication, mitotic abnormalities were at the same level of untreated samples, suggesting a transient effect due to ultrasound. Beside abnormalities of the mitotic spindle, we also observed an increase of metaphases with nonaligned chromosomes. The mitotic index of fibroblasts and HeLa cells, two hours after sonication, showed an intensity-dependent decrease; this was not observed in MCF-7 cells. In agreement with this last result, ultrasound-induced growth inhibition (which was also intensity-dependent) was more marked in fibroblasts and HeLa cells compared to MCF-7 cells. This work indicates that therapeutic ultrasound, even at intensities below the cavitation threshold, can affect genome integrity, showing the need to increase the knowledge of the potential risks of ultrasound to human health. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Very low intensity ultrasounds as a new strategy to improve selective delivery of nanoparticles-complexes in cancer cells.

BACKGROUND: The possibility to combine Low Intensity UltraSound (LIUS) and Nanoparticles (NP) could represent a promising strategy for drugs delivery in tumors difficult to treat overcoming resistance to therapies. On one side the NP can carry drugs that specifically target the tumors on the other the LIUS can facilitate and direct the delivery to the tumor cells. In this study, we investigated whether Very Low Intensity UltraSound (VLIUS), at intensities lower than 120 mW/cm2, might constitute a novel strategy to improve delivery to tumor cells. Thus, in order to verify the efficacy of this novel modality in terms of increase selective uptake in tumoral cells and translate speedily in clinical practice, we investigated VLIUS in three different in vitro experimental tumor models and normal cells adopting three different therapeutic strategies. METHODS: VLIUS at different intensities and exposure time were applied to tumor and normal cells to evaluate the efficiency in uptake of labeled human ferritin (HFt)-based NP, the delivery of NP complexed Firefly luciferase reported gene (lipoplex-LUC), and the tumor-killing of chemotherapeutic agent. RESULTS: Specifically, we found that specific VLIUS intensity (120 mW/cm2) increases tumor cell uptake of HFt-based NPs at specific concentration (0.5 mg/ml). Similarly, VLIUS treatments increase significantly tumor cells delivery of lipoplex-LUC cargos. Furthermore, of interest, VLIUS increases tumor killing of chemotherapy drug trabectedin in a time dependent fashion. Noteworthy, VLIUS treatments are well tolerated in normal cells with not significant effects on cell survival, NPs delivery and drug-induced toxicity, suggesting a tumor specific fashion. CONCLUSIONS: Our data shed novel lights on the potential application of VLIUS for the design and development of novel therapeutic strategies aiming to efficiently deliver NP loaded cargos or anticancer drugs into more aggressive and unresponsive tumors niche.

Genomic damage induced by 1-MHz ultrasound in vitro.

Genotoxic effects of therapeutic ultrasound are poorly documented, when compared with the wide use of this physical agent. The aim of this work was to investigate the clastogenic and aneugenic potential of 1 MHz ultrasound, employing intensities (200 and 300 mW/cm2 ) above the cavitational threshold, but in the range of those normally used in therapeutics. Both normal fibroblasts (AG01522) and tumoral cells (MCF-7) were sonicated. While no effects on viability were noted, significant increases of CREST-negative micronuclei (indicative of clastogenesis) and CREST-positive micronuclei (indicative of aneuploidy) were detected. Clastogenesis was confirmed by increases of γ-H2AX foci, while increases of spindle anomalies confirmed the induction of aneuploidy. Our results confirm previous works that showed ultrasound-induced DNA breakage. Moreover, our experiments show that the known effect of ultrasound-induced damage to microtubules is also able to damage the mitotic spindle and induce aneuploidy. On the overall, this work highlights the importance to further investigate the potential risks related to therapeutics US. Environ. Mol. Mutagen. 59:60-68, 2018. © 2017 Wiley Periodicals, Inc.

Development of a laboratory technique for the evaluation of protease enzymes activity in goat and sheep milk.

The shelf life of milk and its products is strongly influenced by the quality of raw milk. Several bacteria present in raw milk can produce proteases: among them, those from psychotropic microorganisms (e.g. Bacillus subtilis), which produce enzymes under refrigeration, cause the most serious problem. The interaction of these enzymes with the milk proteins and the main proteolytic enzymes becomes important in determining the quality of the milk. An artificial substrate (azocasein) was used to quantify the enzyme activity through the release of a chromogenic product that was measured spectrophotometrically after clarifying the sample by the addition of trichloroacetic acid. The detection limit and quantification limit were 1.43 and 4.77mg/ml respectively for sheep milk and, 2.25 and 7.5mg/ml respectively for goat milk. This method is useful for determining the proteolytic activity in different media and its effectiveness depends on chemical-nutritional characteristics of the sample.

Ultraweak Photon Emission from the Seed Coat in Response to Temperature and Humidity-A Potential Mechanism for Environmental Signal Transduction in the Soil Seed Bank.

Seeds beneath the soil sense the changing environment to time germination and seedling emergence with the optimum time of year for survival. Environmental signals first impact with the seed at the seed coat. To investigate whether seed coats have a role in environmental sensing we investigated their ultraweak photon emission (UPE) under the variable temperature, relative humidity and oxygen conditions they could experience in the soil seed bank. Using a custom-built luminometer we measured UPE intensity and spectra (300-700 nm) from Phaseolus vulgaris seeds, seed coats and cotyledons. UPE was greatest from the internal surface of the seed coat. Seed coat UPE increased concomitantly with both increasing temperature and decreasing relative humidity. Emission was oxygen dependent and it was abolished by treatment with dinitrophenylhydrazine, demonstrating the key role of seed coat carbonyls in the phenomenon. We hypothesize that beneath the soil surface the attenuation of light (virtual darkness: low background noise) enables seeds to exploit UPE for transducing key environmental variables in the soil (temperature, humidity and oxygen) to inform them of seasonal and local temperature patterns. Overall, seed coats were found to have potential as effective transducers of key fluctuating environmental variables in the soil.

Potential genotoxic effects of low-intensity ultrasound on fibroblasts, evaluated with the cytokinesis-block micronucleus assay.

Although medical ultrasound offers compelling opportunities to improve therapy in principle, progress in the field has been limited because of an insufficient understanding of the potential genotoxic and cytotoxic effects of ultrasound on biological systems. This paper is mainly focused on an in vitro study of effects with respect to genotoxicity and viability induced by 1- and 3-MHz medical ultrasound in murine fibroblasts (NIH-3T3) at low-intensity exposure (spatial peak temporal average intensity Ita<0.1 W/cm(2)). The NIH-3T3 cells constitute a well-characterized in vitro cell model in which a genotoxic effect can be predicted by means of a reliable and precise murine cytokinesis-block micronucleus assay. A statistically significant increase in the incidence of micronuclei was observed in sonicated 3T3 cells. In particular, the effects were more evident at 1 MHz. Moreover, for each frequency investigated, the occurrence of micronuclei was comparatively more frequent with increasing time of exposure. The possible toxicological implications of the medical ultrasound employed herein deal with the existence of a window of exposure parameters (set well below the intensity of ultrasound cavitation) in which some genotoxic effects may occur without significant cytotoxicity. In this respect, they provide new insight toward the correct risk to benefit balancing of ultrasound-based treatments and for designing innovative therapeutic strategies.

Structural and permeability sensitivity of cells to low intensity ultrasound: Infrared and fluorescence evidence in vitro.

This work is focused on the in vitro study of the effects induced by medical ultrasound (US) in murine fibroblast cells (NIH-3T3) at a low-intensity of exposure (spatial peak temporal average intensity Ita<0.1Wcm(-2)). Conventional 1MHz and 3MHz US devices of therapeutic relevance were employed with varying intensity and exposure time parameters. In this framework, upon cells exposure to US, structural changes at the molecular level were evaluated by infrared spectroscopy; alterations in plasma membrane permeability were monitored in terms of uptake efficiency of small cell-impermeable model drug molecules, as measured by fluorescence microscopy and flow cytometry. The results were related to the cell viability and combined with the statistical PCA analysis, confirming that NIH-3T3 cells are sensitive to therapeutic US, mainly at 1MHz, with time-dependent increases in both efficiency of uptake, recovery of wild-type membrane permeability, and the size of molecules entering 3T3. On the contrary, the exposures from US equipment at 3MHz show uptakes comparable with untreated samples.

Ultrasound well below the intensity threshold of cavitation can promote efficient uptake of small drug model molecules in fibroblast cells.

Ultrasound (US) induced enhancement of plasma membrane permeability is a hugely promising tool for delivering exogenous vectors at the specific biological site in a safe and efficient way. In this respect, here we report effects of membrane permeability alteration on fibroblast-like cells undergoing very low-intensity of US. The change in permeability was pointed out in terms of high uptake efficiency of the fluoroprobe calcein, thus resembling internalization of small cell-impermeable model drugs, as measured by fluorescence microscopy and flow cytometry. Fluorescence evidences moreover suggests that the higher the time of exposure, the larger will be the size of molecules can be internalized. The uptake events were related to the cell viability and also with structural changes occurring at membrane level as revealed by infrared spectroscopy and preliminary membrane fluidity and atomic force microscopy (AFM) investigation. Thus, the question of whether the uptake of cell-impermeable molecules is consistent with the presence of disruptions on the cell membrane (sonopore formation) has been addressed. In this framework, our findings may constitute experimental evidence in support of sub-cavitation sonoporation models recently proposed, and they may also provide some hints towards the actual working condition of medical US dealing with the optimum risk to benefit therapeutic ratio.

Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: a contribution to the optimization of gene and drug delivery.

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

Metamorphosis delay in Xenopus laevis (Daudin) tadpoles exposed to a 50 Hz weak magnetic field.

PURPOSE: The experiment was performed to prove that exposure to a relatively weak extremely low frequency (ELF) magnetic field retards tadpoles' development. METHODS: Two cohorts of Xenopus laevis laevis (Daudin) tadpoles were exposed during their immature period ( approximately 60 days) to a 50 Hz magnetic field of 63.9 < or = B < or = 76.4 microT rms (root mean square, average values) magnetic flux density in a solenoid. At the same time, as controls, two comparable cohorts were reared in two aquariums remote from the solenoid. Cohorts' degree of development was quantified by daily inspections of animal limbs and attributing them to a stage of the Nieuwkoop and Faber ( 1956 ) classification. The experiment was replicated three times. RESULTS: (a) Mean developmental rate of exposed cohorts was reduced with respect to controls (0.43 vs. 0.48 stages/day, p < 0.001) starting from early larval stages; (b) Exposure increased the mean metamorphosis period of tadpoles by 2.4 days compared with the controls (p < 0.001); (c) Maturation rates of exposed and control tadpoles changed during maturation period; and (d) Important mortality, malformations or teratogenic effects were not observed in exposed matured tadpoles. CONCLUSION: A long-term exposure of X. laevis tadpoles to a relatively weak 50 Hz magnetic field causes a sub-lethal effect that slows down their larval developmental rate and delays their metamorphosis.

Monitoring of people and workers exposure to the electric, magnetic and electromagnetic fields in an Italian National Cancer Institute.

BACKGROUND: The paper reports the electric, magnetic and electromagnetic fields (emf) measurements carried out in the Regina Elena National Cancer Institute (NCI). Several devices, used in diagnostics and in medical cures, can represent sources of emf for the workers and for the public subjected to the treatments. The aim is to evaluate their exposition, in order to assess the compliance with the law. METHODS: The investigations have been carried out in the departments of: intensive care, physiotherapy, MR presstherapy and in the surgical rooms. The measurements have been performed using broad band probes in the frequency ranges 5 Hz/30 kHz and 100 kHz-3 GHz. RESULTS: The variability of the magnetic induction (B(microT)) levels is between 0,05 microT and 80 microT. The statistical distribution shows that most of the measurements are in the range 0,05