Mutagenicity and Antimutagenicity of Baccharis dracunculifolia Extract in Chromosomal Aberration Assays in Chinese Hamster Ovary Cells

Baccharis dracunculifolia De Candole (Asteraceae), a native plant from the Brazilian ""cerrado"", is widely used in folk medicine as an anti-inflammatory agent and for the treatment of gastrointestinal diseases. B. dracunculifolia has been described as the most important plant source of propolis in southeastern Brazil, which is called green propolis due to its color. The aim of the present study was to evaluate the mutagenic and antimutagenic effects of the ethyl acetate extract of B. dracunculifolia leaves (Bd-EAE) on Chinese hamster ovary cells. On one hand, the results showed a significant increase in the frequencies of chromosome aberrations at the highest Bd-EAE concentration tested (100 mu g/mL). On the other hand, the lowest Bd-EAE concentration tested (12.5 mu/mL) significantly reduced the chromosome damage induced by the chemotherapeutic agent doxorubicin. The present results indicate that Bd-EAE has the characteristics of a so-called Janus compound, that is, Bd-EAE is mutagenic at higher concentrations, whereas it displays a chemopreventive effect on doxorubicin-induced mutagenicity at lower concentrations. The constituents of B. dracunculifolia responsible for its mutagenic and antimutagenic effects are probably flavonoids and phenylpropanoids, since these compounds can act either as pro-oxidants or as free radical scavengers depending on their concentration.

Effect of the dibenzylbutyrolactone lignan (-)-hinokinin on doxorubicin and methyl methanesulfonate clastogenicity in V79 Chinese hamster lung fibroblasts

The dibenzylbutyrolactone lignan (-)-hinokinin (HK) was obtained by partial synthesis from (-)-cubebin, isolated from the dry seeds of the pepper, Piper cubeba. In view of the trypanocidal activity of HK and its potential as a lead compound for drug development, evaluation of its possible genotoxic activity is required. We have tested HK for possible genotoxicity and evaluated the compound`s effect on the activity of the clastogens doxorubicin (DXR) and methyl methanesulfonate (MMS) in the micronucleus (MN) assay with Chinese hamster lung fibroblast V79 cells. HK alone did not induce MN, at concentrations up to 128 mu M. In combined treatments, HK reduced the frequency of MN induced by MMS. With respect to DXR, HK exerted a protective effect at lower concentrations, but at higher concentrations it potentiated DXR clastogenicity. (C) 2010 Elsevier B.V. All rights reserved.

Cytotoxic and genotoxic effects of tambjamine D, an alkaloid isolated from the nudibranch Tambja eliora, on Chinese hamster lung fibroblasts

Marine organisms have been shown to be potential sources of bioactive compounds with pharmaceutical applications. Previous chemical investigation of the nudibranch Tambja eliora led to the isolation of the alkaloid tambjamine D. Tambjamines have been isolated from marine sources and belong to the family of 4-methoxypyrrolic-derived natural products, which display promising immunosuppressive and cytotoxic properties. Their ability to intercalate DNA and their pro-oxidant activity may be related to some of the biological effects of the 4-methoxypyrrolic alkaloids. The aim of the present investigation was to determine the cytotoxic, pro-oxidant and genotoxic properties of tambjamine D in V79 Chinese hamster lung fibroblast cells. Tambjamine D displayed a potent cytotoxic effect in V79 cells (IC50 1.2 mu g/mL) evaluated by the MTT assay. Based on the MTT result, V79 cells were treated with different concentrations of tambjamine D (0.6. 1.2. 2.4 and 4.8 mu g/mL). After 24 h, tambjamine D reduced the number of viable cells in a concentration-dependent way at all concentrations tested. assessed by the trypan blue dye exclusion test. The hemolytic assay showed that the cytotoxic activity of tambjamine D was not related to membrane disruption (EC50 > 100 mu g/mL). Tambjamine D increased the number of apoptotic cells in a concentration-dependent manner at all concentrations tested according to acridine orange/ethidium bromide staining, showing that the alkaloid cytotoxic effect was related to the induction of apoptosis. MTT reduction was stimulated by tambjamine D, which may indicate the generation of reactive oxygen species. Accordingly, treatment of cells with tambjamine D increased nitrite/nitrate at all concentrations and TBARS production starting at the concentration corresponding to the IC50. Tambjamine D, also, induced DNA strand breaks and increased the micronucleus cell frequency as evaluated by comet and micronucleus tests, respectively, at all concentrations evaluated. showing a genotoxic risk induced by tambjamine D. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Improved Bioprocess with CHO-hTSH Cells on Higher Microcarrier Concentration Provides Higher Overall Biomass and Productivity for rhTSH

Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (A degrees C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 A degrees C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 x 10(6) cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (similar to 80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (similar to 9 x 10(5) cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce similar to 1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.

Evaluation of the genotoxic and antigenotoxic potential of Baccharis dracunculifolia extract on V79 cells by the comet assay

Baccharis dracunculifolia (Asteraceae), the main botanical source of green propolis, is a shrub of the Brazilian `cerrado`. In folk medicine it is used as an anti-inflammatory agent, mainly for the treatment of gastrointestinal diseases. The aim of the present study was to evaluate the genotoxic and antigenotoxic effects of B. dracunculifolia ethyl acetate extract (Bd-EAE) on Chinese hamster lung fibroblasts (V79 cells) by the comet assay. Methyl methanesulfonate (MMS; 200 mu M) was used as an inducer of DNA damage. Genotoxicity was evaluated using four different concentrations of Bd-EAE: 12.5, 25.0, 50.0 and 100.0 mu g ml(-1). Antigenotoxicity was assessed before, simultaneously, and after treatment with the mutagen. The results showed a significant increase in the frequency of DNA damage in cultures treated with 50.0 and 100.0 mu g ml(-1) Bd-EAE. Regarding its antigenotoxic potential, Bd-EAE reduced the frequency of DNA damage induced by MMS. However, this chemopreventive activity depended on the concentrations and treatment regimens used. The antioxidant activity of phenolic components present in Bd-EAE may contribute to reduce the alkylation damage induced by MMS. In conclusion, our findings confirmed the chemopreventive activity of Bd-EAE and showed that this effect occurs under different mechanism. Copyright (C) 2009 John Wiley & Sons, Ltd.

Continuous and High-Level In Vivo Delivery of Endostatin From Recombinant Cells Encapsulated in TheraCyte (R) Immunoisolation Devices

Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte (R) system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte (R) devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte (R) were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 mu g/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 mu g/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it, demonstrating that ES, secreted by the confined recombinant cells, permeated through the membrane and reached the surrounding tissues.

3`3-Ditrifluoromethyldiphenyl diselenide: A new organoselenium compound with interesting antigenotoxic and antimutagenic activities

The trace element selenium (Se), once known only for its potential toxicity, is now a well-established essential micronutrient for mammals. The organoselenium compound diphenyl diselenide (DPDS) has shown interesting antioxidant and neuroprotective activities. On the other hand, this compound has also presented pro-oxidant and mutagenic effects. The compound 3`3-ditrifluoromethyldiphenyl diselenide (DFDD), a structural analog of diphenyl diselenide, has proven antipsychotic activity in mice. Nevertheless, as opposed to DPDS, little is known on the biological and toxicological properties of DFDD. In the present study, we report the genotoxic effects of the organoselenium compound DFDD on Salmonella typhimurium, Saccharomyces cerevisiae and Chinese hamster lung fibroblasts (V79 cells). DFDD protective effects against hydrogen peroxide (H(2)O(2))-induced DNA damage in vitro are demonstrated. DFDD did not cause mutagenic effects on S. typhimurium or S. cerevisiae strains; however, it induced DNA damage in V79 cells at doses higher than 25 mu M, as detected by comet assay. DFDD protected S. typhimurium and S. cerevisiae against H(2)O(2)-induced mutagenicity, and, at doses lower than 12.5 mu M, prevented H(2)O(2)-induced genotoxicity in V79 cells. The in vitro assays demonstrated that DFDD mimics catalase activity better than DPDS, but neither presents Superoxide dismutase action. The products of the reactions of DFDD or DPDS with H(2)O(2) were different. as determined by electrospray mass spectrometry analysis (ESI-MS). These results suggest that DFDD is not mutagenic for bacteria or yeast; however, it may induce weak genotoxic effects on mammalian cells. In addition, DFDD has a protective effect against H(2)O(2)-induced damage probably by mimicking catalase activity, and the distinct products of the reaction DFDD with H(2)O(2) probably have a fundamental role in the protective effects of DFDD. (C) 2009 Elsevier B.V. All rights reserved.

Cytotoxic and mutagenic evaluation of extracts from plant species of the Miconia genus and their influence on doxorubicin-induced mutagenicity: An in vitro analysis

The Miconia genus, a plant widely used for medicine, occurs in tropical America and its extracts and isolated compounds have demonstrated antibiotic, antitumoral, analgesic and antimalarial activities. However, no study concerning its genotoxicity has been conducted and it is necessary to determine its potential mutagenic effects to develop products and chemicals from these extracts. This study assessed the cytotoxicity, mutagenicity and the protective effects of methanolic extracts from Miconia species on Chinese hamster lung fibroblast cell cultures (V79). The cytotoxicity was evaluated using a clonogenic assay. Cultures exposed to the extract of Miconia albicans up to a concentration of 30 mu g/mL, M. cabucu up to 40 mu g/mL, M. albicans up to 40 mu g/mL and M. stenostachya up to 60 mu g/mL exhibited a cytotoxic effect on the cells. The clonogenic assay used three non-cytotoxic concentrations (5, 10 and 20 mu g/mL) to evaluate mutagenicity and antimutagenicity of the extracts. Cultures were treated with these three extract concentrations (mutagenicity test) or the extract associated with doxorubicin (DXR) (antimutagenicity test) in three protocols (pre-, simultaneous and post-treatments). Distilled water and DXR were used as negative and positive controls, respectively. In the micronucleus (MN) test, a significant reduction was observed in MN frequency in cultures treated with DXR and extracts compared to those receiving only DXR; a significant reduction was also observed for the presence of mutagenicity in all treatments. This study confirmed the safe use of Miconia extracts at the concentrations tested and reinforced the therapeutic properties previously described for Miconia species by showing their protective effects on doxorubicin-induced mutagenicity. (C) 2010 Elsevier GmbH. All rights reserved.

Angiotensin II Binding to Angiotensin I-Converting Enzyme Triggers Calcium Signaling

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system. (Hypertension. 2011;57:965-972.) . Online Data Supplement

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher`s disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

Genetic diversity and toxic activity of Aggregatibacter actinomycetemcomitans isolates

Introduction: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes. Methods: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB. Results: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells. Conclusion: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.

Induction of mitotic crossing-over in diploid strains of Aspergillus nidulans using low-dose X-rays

As a contribution towards detecting the genetic effects of low doses of genotoxic physical agents, this paper deals with the consequences of low-dose X-rays in the Aspergillus nidulans genome. The irradiation doses studied were those commonly used in dental clinics (1-5 cGy). Even very low doses promoted increased mitotic crossing-over frequencies in diploid strains heterozygous for several genetic markers including the ones involved in DNA repair and recombination mechanisms. Genetic markers of several heterozygous strains were individu`ally analyzed disclosing that some markers were especially sensitive to the treatments. These markers should be chosen as bio-indicators in the homozygotization index assay to better detect the recombinogenic/carcinogenic genomic effects of low-dose X-rays.

Cytotoxicity and genotoxicity of bovine pericardium preserved in glycerol

Bovine pericardium is a widely utilized biomaterial. Usually, after harvesting, it is advantageous that the pericardium be immersed in glycerol to improve its shelf life. This can induce some degree of toxicity in the material. The studies were performed in compliance with the rules of ISO 10993 and OECD 487, in the biological evaluation of medical devices. The material was prepared without previous washing. After sterilization by gamma radiation the pericardium was immersed in RPMI 1640 culture medium to fulfill the extraction condition. The same extract was employed in the cytotoxic and genotoxic tests. The procedures were carried out with Chinese hamster ovary cell line and to determine the cytotoxicity, a colorimetric method with the tetrazolium compound MTS was used. For the genotoxicity, following the in vitro micronucleus assay, the test was developed with and without metabolic activation. The Cytotoxicity Index was graphically estimated at the extract concentration of 78%. In the genotoxicity test, the average value of cell proliferation index was found to be 1.62 +/- 0.02 with S9 metabolic activator and 1.91 +/- 0.01 without S9 metabolic activator. Both values are similar to the negative control value in the micronucleus assay. We observed that although the pericardium preserved in glycerol shows a certain level of cytotoxicity, it does not show any genotoxicity.

Antimicrobial and Cytotoxic Activity of Fruit Extract from Syzygium cumini (L.) Skeels

The aim of the present study was to evaluate the antimicrobial and cytotoxic activity of the ethanolic extract of S. cumini according to the Clinical and Laboratory Standards Institute reference method (with modifications), determining the minimal inhibitory and lethal concentration. Activity against Gram-positive (Staphylococcus aureus and S. epidermidis), Gram-negative (Pseudomonas aeruginosa) and yeast of Candida sp and Cryptococcus neoformans was evaluated. The effects of the fruit extract were examined in hamster cells ovaries in concentrations ranging from 1250.0 a 4.9 mu g/ml, measuring the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium. The extract showed both bactericidal and fungicidal activity among the various microorganisms tested and the MIC ranging from 7.8 to 250 mu g/ml. The MIC, MBC and MFC should values that were similar for all the microorganisms. Cytotoxicity index of the dried extract corresponded to the concentration of 400 mu g/ml. The extract could potentially be used in topical antimicrobial products. Thus, the activity of extract was potent to bacteria and mainly to non-albicans species and C. neoformans.

Resistance to ultraviolet-induced apoptosis in DNA repair deficient growth arrested human fibroblasts is not related to recovery from RNA transcription blockage

The impact of ultraviolet (UV-C) photoproducts on apoptosis induction was investigated in growth arrested (confluent) and proliferating human primary fibroblasts. Confluent fibroblasts were more resistant to UV-C-induced apoptosis than proliferating cells, and this was observed for normal human cells and for cells from patients with Cockayne and trichothiodystrophy syndromes, deficient in transcription coupled repair. This resistance was sustained for at least seven days and was not due to DNA repair efficiency, as the removal of CPDs in the genome was similar under both growth conditions. There was no correlation between reduced apoptosis and RNA synthesis recovery. Following UV-C treatment, proliferating and confluent fibroblasts showed a similar level of RNA synthesis inhibition and recovery from transcription blockage. These results support the hypothesis that the decrease of DNA replication, in growth arrested cells, protects cell from UV-C-induced apoptosis, even in the presence of DNA lesions. (C) 2007 Elsevier B.V. All rights reserved.