Photodisinfection of Alphaherpesvirus 1 in bovine semen.

Reproductive biotechnologies are widely consolidated as a methodology in cattle breeding and have an important impact on the genetic improvement of cattle herds. Semen is an important source of dissemination of pathogenic microorganisms during reproductive procedures. To ensure the sanitary quality of the semen, it is essential to consider the presence of various microorganisms including viruses. One of the main viral agents of reproductive interest is Bovine Alphaherpesvirus 1 (BoHV-1), the etiological agent responsible for bovine rhinotracheitis and vulvovaginitis and frequently associated with reproductive efficiency of matrices and bulls. In artificial insemination centers, semen treatment is generally based only on the use of antibiotics, ignoring the possibility of inactivating other non-bacterial infectious agents. In this context, photodisinfection emerges as a promising alternative to inactivate a wide range of microorganisms, offering a complementary or substitution approach to those conventional semen treatment methods. In this work, we evaluated the use of four halogenated sulfonated porphyrins as potential photosensitizers (PSs) for photodynamic inactivation of Bovine Alphaherpesvirus I (BoHV-1) for bovine semen disinfection. The PSs were synthesized and photophysical parameters, such as UV-Vis absorption spectra and singlet oxygen quantum yield (ΦΔ) were presented. Photoinactivation of BoHV-1 was first shown in cell culture and then confirmed in artificially infected bovine semen and then the phototoxicity of PSs against spermatozoa was evaluated. All PSs were effective in BoHV-1 inactivation; however, the photosensitizer containing two chlorine atoms, showed to be more efficient due to the shorter time required for complete viral inactivation. The slight alterations in sperm kinetics were observed, but remained within those acceptable by regulatory agencies for animal reproduction. Although the methodology used in this work only included bovine semen, we emphasize that the proposed photodisinfection methodology can be adapted and applied to a wide range of biological materials and microorganisms of animal or human interest.

Dioclea violacea lectin inhibits tumorigenesis and tumor angiogenesis in vivo.

Dioclea violacea seed mannose-binding lectin (DvL) has attracted considerable attention because of its interesting biological activities, including antitumor, antioxidant, and anti-inflammatory activities. This study evaluated the cytotoxic effect of DvL on tumor and normal cells using the mitochondrial activity reduction (MTT) assay, the carcinogenic and anti-carcinogenic activity by the epithelial tumor test (ETT) in Drosophila melanogaster, and the anti-angiogenic effect by the chick embryo chorioallantoic membrane (CAM) assay. Data demonstrated that DvL promoted strong selective cytotoxicity against tumor cell lines, especially A549 and S180 cells, whereas normal cell lines were weakly affected. Furthermore, DvL did not promote carcinogenesis in D. melanogaster at any concentration tested, but modulated DXR-induced carcinogenesis at the highest concentrations tested. In the CAM and immunohistochemical assays, DvL inhibited sarcoma 180-induced angiogenesis and promoted the reduction of VEGF and TGF-ß levels at all concentrations tested. Therefore, our results demonstrated that DvL is a potent anticancer, anti-angiogenic, and selective cytotoxic agent for tumor cells, suggesting its potential application as a prototype molecule for the development of new drugs with chemoprotective and/or antitumor effects.

Carbon nanotubes and nanofibers seen as emerging threat to fish: Historical review and trends.

Since the discovery of the third allotropic carbon form, carbon-based one-dimensional nanomaterials (1D-CNMs) became an attractive and new technology with different applications that range from electronics to biomedical and environmental technologies. Despite their broad application, data on environmental risks remain limited. Fish are widely used in ecotoxicological studies and biomonitoring programs. Thus, the aim of the current study was to summarize and critically analyze the literature focused on investigating the bioaccumulation and ecotoxicological impacts of 1D-CNMs (carbon nanotubes and nanofibers) on different fish species. In total, 93 articles were summarized and analyzed by taking into consideration the following aspects: bioaccumulation, trophic transfer, genotoxicity, mutagenicity, organ-specific toxicity, oxidative stress, neurotoxicity and behavioral changes. Results have evidenced that the analyzed studies were mainly carried out with multi-walled carbon nanotubes, which were followed by single-walled nanotubes and nanofibers. Zebrafish (Danio rerio) was the main fish species used as model system. CNMs' ecotoxicity in fish depends on their physicochemical features, functionalization, experimental design (e.g. exposure time, concentration, exposure type), as well as on fish species and developmental stage. CNMs' action mechanism and toxicity in fish are associated with oxidative stress, genotoxicity, hepatotoxicity and cardiotoxicity. Overall, fish are a suitable model system to assess the ecotoxicity of, and the environmental risk posed by, CNMs.

Detection of DNA Damage Induced by Cerium Dioxide Nanoparticles: From Models to Molecular Mechanism Activated.

This chapter will present an original effort to summarize the relevant data about the cyto-genotoxicity induced by cerium dioxide nanoparticles (nanoceria) in physiologically (in vivo and in vitro) relevant models. In this way, this chapter should be extremely useful to everyone who wants to plan their research and publishing their results. Massive application of nanoceria at different fields is increasing year after year, and it is urgent to address and discuss their use and its safety-related issues. Specifically, the nanoceria are being designed for nanomedicine, cosmetics, polishing materials and additives for automotive fuels. Their unique properties include the ability to absorb UV radiation, antioxidant potential and the rapid exchange of valence between Ce4+ and Ce3+ ions associated to oxygen storage. In this chapter, the state of the art regarding the physicochemical properties of nanoceria, nanogenotoxicity detected by in vitro and in vivo systems and the general aspects in the cyto-genotoxic mechanism of nanoceria are summarized. The cyto-genotoxicity will be discussed in terms of evaluations by Comet assay, Micronucleus test, DNA damage response and oxidative stress detected in cell culture systems and in vivo test. We also described the dose dependent cyto-genotoxic effects of nanoceria based on their physical-chemical nature. Paradoxically, these particles have been characterized as either pro-oxidant or anti-oxidant in dependence of microenvironment and physiological conditions such as pH. Finally, this chapter will contribute to point out aspects of the development of new in vitro and in vivo methodologies to detect cyto-genotoxic effects of the nanoceria.

Toxic and Beneficial Potential of Silver Nanoparticles: The Two Sides of the Same Coin.

Nanotechnology has allowed great changes in chemical, biological and physical properties of metals when compared to their bulk counterparts. Within this context, silver nanoparticles (AgNPs) play a major role due to their unique properties, being widely used in daily products such as fabrics, washing machines, water filters, food and medicine. However, AgNPs can enter cells inducing a "Trojan-horse" type mechanism which potentially leads to cellular autophagy, apoptosis or necrosis. On the other hand, this cytotoxicity mechanism can be optimized to develop drug nanocarriers and anticancer therapies. The increasing use of these NPs entails their release into the environment, damaging ecosystems balance and representing a threat to human health. In this context, the possible deleterious effects that these NPs may represent for the biotic and abiotic ecosystems components represent an obstacle that must be overcome in order to guarantee the safety use of their unique properties.

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer.

Light source is critical to induce glioblastoma cell death by photodynamic therapy using chloro-aluminiumphtalocyanine albumin-based nanoparticles.

Selection of an efficient light source is fundamental in the development of photodynamic therapy (PDT) protocols. However, few studies provide a comparison of different light sources with regard to phototoxic effects. Here, we compared the cell death induced by photoactivation of chloro-aluminiumphtalocyanine (AlClPc)-loaded human serum albumin nanoparticles under irradiation with different light sources: continuous laser (CL), pulsed laser (PL), and light-emitting diode (LED). Cells were exposed to three different AlClPc concentrations (1, 3, and 5µM) and three different light doses (200, 500, and 700mJ/cm2) for each light source. Cell death and differentiation of apoptosis and necrosis pathway were measured by flow cytometry. CL was the best light source for improving the photodynamic action of AlClPc-loaded albumin nanoparticles in glioblastoma cells and avoiding undesirable side effects, especially at low photosensitizer doses (200mJ/cm2). In addition, apoptosis was the main cell death pathway in all evaluated cases (70% for CL, and greater than 50% for PL and LED). In conclusion, the search for optimal light sources and light/photosensitizer doses is a crucial step in improving PDT outcomes and enhancing the clinical translation of PDT.

In vitro effects of photodynamic therapy induced by chloroaluminum phthalocyanine nanoemulsion.

BACKGROUND: The photodynamic therapy (PDT) has been used to treat cancer mainly by inducing oxidative stress. Our aim was to evaluate the effect of PDT and its combination with methoxyamine (MX), a blocker of base excision repair (BER), in cells expressing high levels of the APE1 protein, which is involved in cell oxidative damage response. METHODS: The HeLa and A549 cells were treated for 3h with chloroaluminum phthalocyanine incorporated into a well-designed nanoemulsion (ClAlPc/NE); and then irradiated by visible light (@670nm) with doses of 0.1, 0.5 and 1.0J/cm2. A simultaneous combination of MX+ClAlPc/NE was performed and then irradiated with the selected dose of 0.5J/cm2. The treatments were evaluated in terms of viability, clonogenicity, DNA fragmentation, and cell death mechanism by apoptosis and/or necrosis. RESULTS: The APE1 protein expression observed was higher in HeLa than in A549. Both cell lines exhibited substantial differences in cell cytotoxicity. The PDT decreased the clonogenicity of HeLa by inducing apoptosis (sub-G1 and annexin detection). Additionaly, the MX potentiates the PDT-effects in HeLa. Otherwise, low cytotoxicity was observed in A549 cells. CONCLUSION: The PDT induced apoptosis in high APE1 expressive HeLa cells, and the blockage of BER by MX increased its effects.

Evaluation of carbon nanotubes network toxicity in zebrafish (Danio rerio) model.

This is a detailed in vivo study of the biological response to carbon nanotubes network as probed by the zebrafish model. First, we prepared pristine carbon nanotubes (CNTs) by methanol chemical vapor deposition in the presence of Mn and Co as catalysts, followed by purification in acid, which furnished curved tubes with diameters lying between 10 and 130 nm. The CNT network consisted of pristine CNTs dispersed in water in the presence of a surfactant. The CNT network pellets corresponded to agglomerated multi-walled CNTs with an average diameter of about 500 nm. Although the same pristine CNTs had been previously found to exert genotoxic effects in vitro, here we verified that the CNT network was not genotoxic in vivo. Indeed, Raman spectroscopy and microscopy conducted in the intestine of the zebrafish revealed complete clearance of the CNT network as well as minimal disturbances, such as aneurysms, hyperemia, and reversible inflammatory focus in the zebrafish gills.

Cytotoxicity and genotoxicity of coronaridine from Tabernaemontana catharinensis A.DC in a human laryngeal epithelial carcinoma cell line (Hep-2).

Cancer has become a major public health problem worldwide and the number of deaths due to this disease is increasing almost exponentially. In the constant search for new treatments, natural products of plant origin have provided a variety of new compounds to be explored as antitumor agents. Tabernaemontana catharinensis is a medicinal plant that produces alkaloids with expressive antitumor activity, such as heyneanine, coronaridine and voacangine. The aim of present study was firstly to screen the cytotoxic activity of the indole alkaloids heyneanine, coronaridine and voacangine against HeLa (human cervix tumor), 3T3 (normal mouse embryo fibroblasts), Hep-2 (human laryngeal epithelial carcinoma) and B-16 (murine skin) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); and secondly to analyze the apoptotic activity, cell membrane damage and genotoxicity of the compound that showed the best cytotoxic activity against the tumor cell lines tested. Coronaridine was the one that exhibited greater cytotoxic activity in the laryngeal carcinoma cell line Hep-2 (IC50 = 54.47 µg/mL) than the other alkaloids tested (voacangine IC50 = 159.33 g/mL, and heyneanine IC50 = 689.45 µg/mL). Coronaridine induced apoptosis in cell lines 3T3 and Hep-2, even at high concentrations. The evaluation of genotoxicity by comet assay showed further that coronaridine caused minimal DNA damage in the Hep-2 tumor cell line, and the LDH test showed that it did not affect the plasma membrane. These results suggest that further investigation of coronaridine as an antitumor agent has merit.

Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with osteogenic growth peptide for bone tissue engineering.

The aim of this study was to characterize the physicochemical properties of bacterial cellulose (BC) membranes functionalized with osteogenic growth peptide (OGP) and its C-terminal pentapeptide OGP[10-14], and to evaluate in vitro osteoinductive potential in early osteogenesis, besides, to evaluate cytotoxic, genotoxic and/or mutagenic effects. Peptide incorporation into the BC membranes did not change the morphology of BC nanofibers and BC crystallinity pattern. The characterization was complemented by Raman scattering, swelling ratio and mechanical tests. In vitro assays demonstrated no cytotoxic, genotoxic or mutagenic effects for any of the studied BC membranes. Culture with osteogenic cells revealed no difference in cell morphology among all the membranes tested. Cell viability/proliferation, total protein content, alkaline phosphatase activity and mineralization assays indicated that BC-OGP membranes enabled the highest development of the osteoblastic phenotype in vitro. In conclusion, the negative results of cytotoxicity, genotoxicity and mutagenicity indicated that all the membranes can be employed for medical supplies, mainly in bone tissue engineering/regeneration, due to their osteoinductive properties.

Mutagenic and recombinagenic effects of lamivudine and stavudine antiretrovirals in somatic cells of Drosophila melanogaster.

Lamivudine (3TC) and stavudine (d4T) are nucleoside analogue reverse transcriptase inhibitors employed in antiretroviral therapies. The mutational and recombinational potential as well as the total genetic toxicity was determined for both compounds at concentrations allowing at least 30% survival using the standard version of wing SMART assay. The standardized clone induction frequency per mg/ml for mwh/flr(3) genotype were approximately 2 and approximately 33 mutant clones/10(5) cells/(mg/ml) for d4T and 3TC, respectively. Comparing these results with those obtained in the mwh/TM3 genotype, it was possible to quantify the recombinagenic action of each drug. Approximately 86% of the mutant clones induced by 3TC and approximately 76% of the d4T induced clones were related to their mitotic recombination action. Our results indicate that both 3TC and d4T have high recombinagenic potential, and suggest that exposure to the drugs could cause genomic instability and loss of heterozygosity. This may be due to the fact that these genetic alterations play a primary role in carcinogenesis, and are also involved in secondary and subsequent steps of carcinogenesis by which recessive oncogenic mutations are revealed.