Characterization of abalone Haliotis tuberculata–Vibrio harveyi interactions in gill primary cultures

The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell—V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments.     

Effects of Triclosan on Mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possible modes of action on non target organisms

Pharmaceuticals and Personal Care Products (PPCPs) are a class of emerging environmental pollutants with the potential of affecting various aquatic organisms through unexpected modes of action. Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) (TCS), is a common antibacterial agent that is found in significant amounts in the aquatic environment. In this work, the possible effects and modes of action of TCS were investigated in the marine bivalve Mytilus galloprovincialis Lam. In mussel immune cells, the hemocytes, in vitro short-term exposure to TCS in the low microM range reduced lysosomal membrane stability (LMS) and induced extracellular release of lysosomal hydrolytic enzymes. The effects on LMS were mediated by activation of ERK MAPKs (Extracellularly Regulated Mitogen Activated Protein Kinases) and PKC (protein kinase C) alpha and betaII isoforms, as demonstrated by both specific kinase inhibitors and Western blotting with specific anti-phospho-antibodies. The effects of TCS were confirmed in vivo, in the hemocytes of mussels injected with different concentrations of TCS (corresponding to 0.29, 2.9 and 29 ng/g dry weight) and sampled at 24 h post-injection. The possible in vivo effects of TCS were also evaluated on the activity of different enzymes in the digestive gland, the tissue mainly involved in accumulation and metabolism of organic contaminants in mussels. Significant increases were observed in the activity of the glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase), as well as of GST (GSH transferase) and GSR (GSSG reductase), whereas a decrease in catalase activity was observed. The results demonstrate that in mussels TCS can act on kinase-mediated cell signalling, lysosomal membranes and redox balance in different systems/organs. Although further studies are needed in order to evaluate possible consequences of environmental exposure to TCS on mussel health, the results represent the first data on the possible modes of action of this widespread antibacterial in aquatic invertebrates.     

Transformation of triclosan by Trametes versicolor and Pycnoporus cinnabarinus

We investigated the ability of Trametes versicolor and Pycnoporous cinnabarinus to metabolize triclosan. T. versicolor produced three metabolites, 2-O-(2,4,4'-trichlorodiphenyl ether)-beta-D-xylopyranoside, 2-O-(2,4,4'-trichlorodiphenyl ether)-beta-D-glucopyranoside, and 2,4-dichlorophenol. P. cinnabarinus converted triclosan to 2,4, 4'-trichloro-2'-methoxydiphenyl ether and the glucoside conjugate known from T. versicolor. The conjugates showed a distinctly lower cytotoxic and microbicidal activity than triclosan did.     

Forms of Selenium Affect its Transport, Uptake and Glutathione Peroxidase Activity in the Caco-2 Cell Model

The aim of this study was to investigate the possible time- and dose-dependent cytotoxic effects of cobalt chloride on Vero cells. The cultured cells were incubated with different concentrations of cobalt chloride ranging from 0.5 to 1,000 μM, and cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and resazurin assays. Possible protective effects of vitamin E, coenzyme Q(10), and zinc chloride were also tested in this system. A gradual decrease in cell proliferation was observed at concentrations ~≥ 200 μM in incubation periods of 24, 48, 72, and 96 h with MTT assay. Exposure of cells to 500 and 1,000 μM cobalt chloride caused significant decrease in cell survival. A biphasic survival profile of cells was observed at 1-25 μM concentration range following 96 h of incubation. With resazurin assay, cytotoxicity profile of CoCl(2) was found comparable to the results of MTT assay, particularly at high concentrations and long incubation periods. Dose-dependent cytotoxicity was noted following exposure of cells to ≥ 250 μM of CoCl(2) for 24 h and ≥ 100 μM concentrations of CoCl(2) for 48-96 h. Pretreatment of cells with ZnCl(2) for 4 or 24 h provided significant protection against cobalt chloride-induced cytotoxicity when measured with MTT assay. However, vitamin E or coenzyme Q(10) was not protective. CoCl(2) had dose- and time-dependent cytotoxic effects in Vero cells. Preventive effect of ZnCl(2) against CoCl(2)-induced cytotoxicity should be considered in detail to define exact mechanism of toxicity in Vero cells.     

Paradigm shifts in malaria parasite biochemistry and anti-malarial chemotherapy

A fatty acid synthesis (FAS) pathway was recently discovered and established in the obligate human parasite Plasmodium falciparum. Its inhibition by triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) leads to its classification as a type II FAS. Humans, the vertebrate host for the malarial parasite utilize type I FAS, which is not inhibited by triclosan. This discovery thus paves the way for novel approaches to the treatment of malaria. In direct contrast to the delayed-death phenotype associated with poisoning of the apicoplast using certain other drugs, the rapid and striking action of triclosan suggests the possibility of developing new drug(s) for the treatment of malaria.     

Low-density lipoprotein receptor-related protein 1 is an essential receptor for trichosanthin in 2 choriocarcinoma cell lines

Type-I ribosome-inactivating protein-trichosanthin (TCS) exhibits selective cytotoxicity toward different types of cells. It is believed that the cytotoxicity results from the inhibition of ribosomes to decrease protein synthesis, thereby indicating that there are specific mechanisms for TCS entry into target cells to reach the ribosomes. Low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a large scavenger receptor that is responsible for the binding and endocytosis of diverse biological ligands on the cell surface. In this study, we demonstrated that 2 choriocarcinoma cell lines can significantly bind and internalize TCS. In contrast, Hela cell line displayed no obvious TCS binding and endocytosis. Furthermore LRP1 gene silencing in JAR and BeWo cell lines blocked TCS binding; TCS could also interact with LRP1.The results of our study established that LRP1 was a major receptor for phagocytosis of TCS in JAR and BeWo cell lines and might be the molecular basis of TCS abortificient and anti-choriocarcinoma activity.     

Occurrence of Perkinsus olseni in the Venus clam Protothaca jedoensis in Korean waters

This is the first report of the occurrence of Perkinsus olseni in the Venus clam Protothaca jedoensis off the western and southern coasts of South Korea. Histological observations revealed Perkinsus-like organisms in the mantle, gills, digestive tubules, and gonad. Haemocytic infiltration and tissue necrosis were also observed in heavily infected clams. Hypnospore formation of the Perkinsus-like organism was confirmed with Ray's fluid thioglycollate medium assay (RFTM). When incubated in filtered and aerated seawater, the hypnospore gave rise to cell division and subsequently discharged hundreds of motile zoospores. Genus- and species-specific polymerase chain reaction (PCR) assays and the DNA sequences of the internal transcribed spacer region (ITS) of the Perkinsus sp. isolated from the Venus clam were identical to those of P. olseni reported from the Manila clam Venerupis (=Ruditapes)philippinarum. Based on the DNA sequences and microscopic data, the Perkinsus-like pathogen isolated from P. jedoensis was identified as P. olseni, which parasitizes the Manila clam in European and Asian waters and Haliotis rubra (abalone) in Australian waters. The prevalence and infection intensity of a clam population collected from Yosu, Korea, was determined using RFTM and Choi's 2M NaOH digestion technique. The intensities averaged 10,768 and 7438 Perkinsus cells per gram tissue in 2003 and 2004, and the prevalence ranged from 37.0 to 53.9%, respectively.     

Molecular cloning and expression analysis of interferon-gamma inducible lysosomal thiol reductase (GILT)-like cDNA from disk abalone (Haliotis discus discus)

Interferon Gamma (IFN-gamma) Inducible Lysosomal Thiol reductase (GILT) has been described as a key enzyme in processing and presentation of major histocompatibility complex (MHC) class II restricted antigen (Ag) by catalyzing disulfide bond (S-S) reduction in mammals. Abalone GILT-like (AbGILT) full-length cDNA was isolated from the normalized disk abalone cDNA library. The 807-bp AbGILT cDNA consists of an open reading frame of 684-bp, encoding 228 amino acid residues. The predicted AbGILT protein has a molecular weight of 25kDa and an isoelectric point of 7.8. The N-terminus of the AbGILT was found to have a putative signal peptide with a cleavage site amino acid position at 19-20. AbGILT contains two active site C-XX-C motifs, ((23)CLDC(26) and (46)CPYC(49)) which motif is highly conserved in GILT protein family. AbGILT exhibited a characteristic GILT signature sequence (92)CQHGX(2)ECX(2)NX(4)C(107) and 12 cysteine residues representing 5% in the mature peptide. Phylogenetic analysis showed that AbGILT has been derived from a common ancestor with other GILT proteins. RT-PCR results showed that AbGILT expression was up-regulated in the gill, mantle and digestive tract 24h post injection of phytohemagglutinin (PHA) while Vibrio alginolyticus up-regulation appeared in the gill and digestive tract after 48h. In contrast, AbGILT expression was not up-regulated by poly inosinic-cytidylic acid (poly I:C) during the 48h induction. However, AbGILT was constitutively expressed in gill, mantle, and digestive tract tissues suggesting that it may maintain first line of innate immune defense at basal level in disk abalone.     

Pancreatic anticancer activity of a novel geranylgeranylated coumarin derivative

A series of hydroxycoumarin derivatives has been synthesized and evaluated against human pancreatic PANC-1 cancer cells under nutrient-deprived conditions. Several compounds exhibited 100% preferential cytotoxicity at low micromolar concentrations under nutrition starvation, and showed no cytotoxicity under nutrient-rich conditions. In this study, a novel geranylgeranylated ether coumarin derivative 9 was found to exhibit the highest cytotoxic activity of 6.25 μM within 24h. The preferential anti-tumor activity exhibited by compound 9 against PANC-1 under low oxygen and nutrient environment illustrates its great potential as a promising lead structure for the development of novel agents to combat pancreatic cancer.     

Cytotoxic evaluation of sarcodifurines A and B,two novel dihydrofuroquinolines from Sarcomelicope follicularis (Rutaceae)

Sarcodifurines A and B are two original dihydrofuroquinolines isolated from Sarcomelicope follicularis, a New Caledonian tree. The cytotoxicity and antiproliferative activity of these two alkaloids were investigated against 8 distinct cell lines representative of the most frequent solid tumors developing in human. Cytotoxicity of sarcodifurines was low on the 8 cell lines, with, for example, less than 10% of the total cells killed after 24 h exposure at 10 μM and IC₅₀ ≈ 7.10^{? 5} M (MCF-7 and MDA MB 231 cell lines). Proliferation studies confirmed that sarcodifurines had a weak effect on cancer cells growth, with less than 5% growth inhibition at 10 μM. Sarcodifurine A activity was comparable to that of Sarcodifurine B, in term of cytotoxicity and antiproliferative activity on all cell lines. In spite of the weak activity of sarcodifurines and furoquinolines, rationalized pharmacomodulations to obtain planar analogs could lead to efficient topoisomerases inhibitors and DNA intercalants.     

Quercetin both partially attenuates hydrogen peroxide-induced toxicity and decreases viability of rat glial cells

Quercetin is one of the most ubiquitous flavonoids in foods of plant origin. Although quercetin is generally considered to provide protection against oxidative injury, recent studies have shown to be cytotoxic to many cell types. We intended here to determine whether quercetin protects against H2O2-induced toxicity and/or affects viability of rat mixed glial cells. The cells were obtained from 1-3 day olds rat brains and incubated in a humidified atmosphere of 5% CO2, at 37 °C in flasks. In the quercetin groups, different quercetin concentrations (1, 10, 50, 75 or 100 μM) were applied alone for 24 h. For H2O2 cytotoxicity group, the glial cells were treated for 3 h with 100 μM H2O2 which induced 75% cell death. In another group, the cells were treated with 100 μM H2O2 plus respective quercetin concentrations simultaneously for 3 h, the medium was removed and refed for 24 h. MTT test was then applied and statistical significance was ascertained by one way analysis of variance, followed by Tukey's multiple comparison test. Quercetin starting from 50 μM decreased the glia survival significantly. In H2O2 plus quercetin co-treated groups, both 75 and 100 μM quercetin attenuated toxic effect of H2O2 by 15%. In conclusion, quercetin both partially protects H2O2-induced gliotoxicity and decreases rat glial cell viability in primary culture.     

Cytotoxic and genotoxic effects of Br-containing oxaphosphole on Allium cepa L. root tip cells and mouse bone marrow cells

The continuous production and release of chemicals into the environment has led to the need to assess their genotoxicity. Numerous organophosphorus compounds with different structures have been synthesized in recent years, and several oxaphosphole derivatives are known to possess biological activity. Such chemical compounds may influence proliferating cells and cause disturbances of the genetic material. In this study, we examined the cytotoxicity and genotoxicity of 4-bromo-N,N-diethyl-5,5-dimethyl-2,5-dihydro-1,2-oxaphosphol-2-amine 2-oxide (Br-oxph). In A. cepa cells, Br-oxph (10(-9) M, 10 (-6) M and 10 (-3) M) reduced the mitotic index 48 h after treatment with the two highest concentrations, with no significant effect at earlier intervals. Mitotic cells showed abnormalities 24 h and 48 h after treatment with the two lowest concentrations but there were no consistent changes in interphase cells. Bone marrow cells from mice treated with Br-oxph (2.82 x 10 (-3) μg/kg) also showed a reduced mitotic index after 48 h and a greater percentage of cells with aberrations (principally chromatid and isochromatid breaks). These findings indicate the cytotoxicity and genotoxicity of Br-oxph in the two systems studied.     

Synthesis and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methyl-7-deazapurine nucleosides, their phosphoramidate prodrugs and 5'-triphosphates

Thirty novel α- and β-d-2'-deoxy-2'-fluoro-2'-C-methyl-7-deazapurine nucleoside analogs were synthesized and evaluated for in vitro antiviral activity. Several α- and β-7-deazapurine nucleoside analogs exhibited modest anti-HCV activity and cytotoxicity. Four synthesized 7-deazapurine nucleoside phosphoramidate prodrugs (18-21) showed no anti-HCV activity, whereas the nucleoside triphosphates (22-24) demonstrated potent inhibitory effects against both wild-type and S282T mutant HCV polymerases. Cellular pharmacology studies in Huh-7 cells revealed that the 5'-triphosphates were not formed at significant levels from either the nucleoside or the phosphoramidate prodrugs, indicating that insufficient phosphorylation was responsible for the lack of anti-HCV activity. Evaluation of anti-HIV-1 activity revealed that an unusual α-form of 7-carbomethoxyvinyl substituted nucleoside (10) had good anti-HIV-1 activity (EC(50)=0.71±0.25 μM; EC(90)=9.5±3.3 μM) with no observed cytotoxicity up to 100 μM in four different cell lines.     

Some commonly used brominated flame retardants cause Ca2+-ATPase inhibition, beta-amyloid peptide release and apoptosis in SH-SY5Y neuronal cells

Brominated flame retardants (BFRs) are chemicals commonly used to reduce the flammability of consumer products and are considered pollutants since they have become widely dispersed throughout the environment and have also been shown to bio-accumulate within animals and man. This study investigated the cytotoxicity of some of the most commonly used groups of BFRs on SH-SY5Y human neuroblastoma cells. The results showed that of the BFRs tested, hexabromocyclododecane (HBCD), tetrabromobisphenol-A (TBBPA) and decabromodiphenyl ether (DBPE), all are cytotoxic at low micromolar concentrations (LC(50) being 2.7 ± 0.7 μM, 15 ± 4 μM and 28 ± 7 μM, respectively). They induced cell death, at least in part, by apoptosis through activation of caspases. They also increased intracellular [Ca(2+)] levels and reactive-oxygen-species within these neuronal cells. Furthermore, these BFRs also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells. Elevated intracellular [Ca(2+)] levels appear to occur through a mechanism involving microsomal Ca(2+)-ATPase inhibition and this maybe responsible for Ca(2+)-induced mitochondrial dysfunction. In addition, μM levels of these BFRs caused β-amyloid peptide (Aβ-42) processing and release from these cells with a few hours of exposure. These results therefore shows that these pollutants are both neurotoxic and amyloidogenic in-vitro.     

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 g l⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2′-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT₅₀/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC₅₀. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.     

Characterization of binding sites for calcitonin gene related peptide in abalone gill.

Target organs for calcitonin gene related peptide were investigated in the abalone. To elucidate the function of this neuropeptide in the biomineralization process, we have localized, in different tissues from abalone, specific binding sites for human calcitonin gene related peptide (hCGRP). Highest binding was observed in gill membranes where two classes of affinity components were identified. The affinity constants and the number of binding sites per mg of proteins for the site I were 5 X 10(9)M(-1)and 8.8 x 10(10). For the site II, the affinity constant was 1.34 X 10(7) M(-1)and the number of binding sites per mg of proteins was 1.1 x 10(12). In contrast, no specific calcitonin binding could be detected in every tested tissue, but the similar displacement of the 125I-labeled CGRP binding with unlabeled hCGRP and sCT suggested that, in the abalone, the identified receptor could belong to a third class receptor subtype, that recognize both hCGRPalpha and sCT. These data suggest that, during evolution, the role of CGRP in gill function is particularly well conserved and that this neuropeptide is likely to participate in the control of hydromineral metabolism in aquatic species.     

Synthesis of 6-substituted 9-methoxy-11H-indeno[1,2-c]quinoline-11-one derivatives as potential anticancer agents

We have synthesized certain 6-substituted 9-methoxy-11H-indeno[1,2-c]quinolin-11-ones for antiproliferative evaluation. Results indicated that 6-alkylamine derivatives, 6-[2-(dimethylamino)ethylamino]-9-methoxy-11H-indeno[1,2-c]quinolin-11-one (5a) and its 6-[3-(dimethylamino)propylamino] derivative, 5b, were able to inhibit cells growth completely at a concentration of 100 μM while most of the 6-arylamine derivatives 6b-6h were inactive at the same concentration. Comparable mean GI(50) (drug molar concentration causing 50% cell growth inhibition) values for 5a (3.47 μM) and 5b (3.39 μM) indicated the cytotoxicity may not be affected by the length of alkyl substituents at C-6 position. Compound 5b, with a mean GI(50) value of 3.39 μM, was the most active and therefore was selected for further evaluation on its effects of H460 lung cancer cell cycle distribution. Results indicated that 5b induced cell cycle arrest in G2/M phase after 24h treatment, while the hypodiploid (sub-G0/G1 phase) cells increased. We found that H460 cell became shrinked after the treatment of 5b, indicating that apoptosis may be a mechanism by which 5b kills the cancer cells. DNA unwinding assay indicated that 5b may bind to DNA through intercalation. Our results have also demonstrated that PARP was cleaved while caspase-3 and caspase-8 activities were induced after the treatment of 5b at 10 μM for 24h. Thus, compound 5b intercalates DNA, induces cell cycle arrest at G2/M phase via cleavage of PARP, induces caspase-3 and caspase-8 activities, and consequently causes the cell death.     

Effect of Fe-Pd bimetallic nanoparticles on Sphingomonas sp. PH-07 and a nano-bio hybrid process for triclosan degradation

In this study, we have evaluated the effect of palladium-iron bimetallic nanoparticles (nFe-Pd) on diphenyl ether (DE) degrading bacterial strain Sphingomonas sp. PH-07 as well as a sequential nano-bio hybrid process with nFe-Pd as catalytic reductant and PH-07 as biocatalyst for degradation of triclosan. Strain PH-07 grew well in the presence of nFe-Pd up to 0.1 g/L in minimal salts medium with DE as carbon source. In aqueous system, TCS (17.3 μM) was completely dechlorinated within 2 h by nFe-Pd (0.1 g/L) with concomitant release of 2-phenoxyphenol (16.8 μM) and chloride ions (46 μM). All possible dichloro- and monochloro-2-phenoxyphenol intermediates were identified by HPLC and GC-MS analyses, and the dechlorination pathway was proposed. Addition of PH-07 cells into the reactor effectively degraded the 2-phenoxyphenol. Our results reveal that strain PH-07 survives well in the presence of nFe-Pd and nFe-Pd/PH-07 hybrid treatment could be a potential strategy for degradation of TCS.     

Indole alkaloids from Muntafara sessilifolia with antiplasmodial and cytotoxic activities

Four vobasinyl-iboga bisindole and one 2-acyl monomeric indole alkaloids were isolated from the stem bark of Muntafara sessilifolia along with eleven known compounds. Their structures and relative stereochemistry were elucidated on the basis of spectroscopic data including 1D and 2D NMR and mass spectrometry (MS). All isolated compounds were evaluated in vitro for antiplasmodial activity against the chloroquine-resistant strain FcB1 of Plasmodium falciparum, and for cytotoxicity against the human lung cell line MRC-5 and the rat skeletal muscle cell line L-6. 3'-Oxo-tabernaelegantine A exhibited antiplasmodial activity (4.4 μM IC(50)) associated with non-significant cytotoxicity (selectivity index of 48). Tabernaelegantine B and D displayed the highest cytotoxicity with IC(50) values of 0.47 and 1.89 μM on MRC-5 cells, and 0.42 and 2.7 μM on L-6 cells, respectively.