Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin

The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (Cl) = 111-130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5-3.0 microM (200-1200 micrograms/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r2 = 0.99) and HPLC (r2 = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts.     

Inhibition of protein kinase C activity by the antirheumatic drug auranofin

The Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C; PKC) has a central role in the transmission of extracellular signals. The orally active anti-rheumatic drug, auranofin, has been shown to modulate PKC-mediated cell responses. In this study, we report that auranofin directly inhibits PKC in a dose-dependent manner; inhibition can be overcome by mercapto-ethanol. Proteolytically-activated PKC is also inhibited by auranofin excluding an effect of the drug on the regulatory domain of the enzyme. These data clearly show that auranofin inhibits the catalytic activity of PKC, probably by interacting with thiol groups.     

Expression of Drosophila Ca2+ permeable transient receptor potential-like channel protein in a prostate cancer cell line decreases cell survival

The effects of expression of Drosophila melanoga ster Ca(2+) permeable transient receptor potential-like (TRPL) channels, under the control of the cytomegalovirus (CMV) or prostate cell-specific promoters, on cell survival and apoptosis in the androgen-sensitive LNCaP prostate cancer cell line were investigated. A prostate-specific antigen (PSA) promoter construct (designated PSAEn/PSAPr) composed of a 0.6 kb region of the promoter and a 1.45 kb region of the enhancer resulted in androgen-dependent and prostate-specific expression of a luciferase reporter gene in transiently transfected LNCaP cells. Expression of the enhanced green fluorescence protein-TRPL chimeric protein under the control of the CMV promoter was confirmed by Western blot. Whereas the majority of the expressed protein was located in the cytoplasmic space, confocal microscopy with the CD-9 protein as a plasma membrane marker demonstrated that approximately 10% of the expressed TRPL protein was located in a band in the plasma membrane. Using recombinant adenoviruses, expression of the TRPL protein was associated with an increase in both the initial and sustained rates of Ca(2+) inflow. Expression of TRPL under the control of the CMV promoter for 96 hours decreased cell number and increased the number of cells undergoing apoptosis by 23 and 27%, respectively. Apoptosis was inhibited by a caspase-3 inhibitor, Z-DEVD-fmk. It is concluded that, when heterologously expressed in LNCaP cells, the TRPL protein leads to a reduction in cell survival due, in part, to the induction of apoptosis. The effects of TRPL are likely caused by enhanced Na(+) and Ca(2+) inflow to the cells. This finding suggests a novel approach to modify the growth of prostate cancer cells that fail to undergo apoptosis following androgen ablation therapy.     

Effect of tumor promoters on the activity of cyclic adenosine 3':5'-monophosphate-dependent and -independent protein kinases from mouse epidermis.

Cyclic adenosine 3'5'-monophosphate (cyclic AMP)-dependent and -independent protein kinases were detected and partially characterized in soluble extracts from mouse epidermis. Cylic AMP-dependent histone kinase activity was separated rom cyclic AMP-independent casein kinase activity by DEAE-Sephadex chromatography. The application of the tumor promoters croton oil or 12-o-tetradecanoyl-phorbol-13-acetate to mouse skin caused a rapid increase in the soluble protein extractable from the epidermis resulting in a decrease in the specific activity of both classes of protein kinase when expressed on a protein basis. No change in the activities of either the cyclic AMP-dependent or -independent enzymes was observed when expressed relative to the DNA content.     

An examination of the antibiotic effects of cylindrospermopsin on common gram-positive and gram-negative bacteria and the protozoan Naegleria lovaniensis

The importance of the toxin cylindrospermopsin to the function and fitness of the cyanobacteria that produce it remains a matter of conjecture. Given that the structure of cylindrospermopsin has commonalities with other antibacterial protein synthesis inhibitors, such as streptomycin, authors tested the possibility that the toxin might act as an antibacterial compound that can kill competing microbes. Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa were tested by the minimal inhibitory concentration method and significant antibacterial activity was only observed at a cylindrospermopsin concentration of 300 microg mL(-1) after exposure for 5 days. No effect on log phase growth of E. coli was observed for this same toxin concentration. Protein synthesis was inhibited by cylindrospermopsin in E. coli 70S extracts, reduced by 25% compared with controls when treated with 41.5 microg mL(-1) of the toxin; however, a much greater reduction of 97% was observed for chloramphenicol in the same experiment. Naegleria lovaniensis, a phagotrophic protozoan, was more susceptible to cylindrospermopsin, with a decrease in the number of N. lovaniensis plaques after 24-h treatment with 5-50 microg mL(-1) of toxin and an LC(50) of approximately 60 microg mL(-1). Given these results, cylindrospermopsin is clearly not antibacterial at concentrations found in environmental waters, nor will it adversely affect N. lovaniensis at these concentrations. For organisms that are able to ingest cylindrospermopsin-producing cells, the response of N. lovaniensis to the toxin suggests that only a few ingested cells would be enough to kill predatory organisms with similar susceptibility.     

Localization of adenosine o',5'-monophosphate in mouse epidermis by immunofluorescence.

Adenosine 3',5'-monophosphate (cyclic AMP) has been localized in mouse epidermal cells using an immunofluorescent technique. Within 10 min following the intraperitoneal injection of isoproterenol or 30 hr following the topical application of croton oil in acetone, staining was clearly visible in the cytoplasm of the basal cells.     

Cylindrospermopsin genotoxicity and cytotoxicity: role of cytochrome P-450 and oxidative stress

Cylindrospermopsin (CYN) is a cyanobacterial toxin found in drinking-water sources world wide. It was the likely cause of human poisonings in Australia and possibly Brazil. Although CYN itself is a potent protein synthesis inhibitor, its acute toxicity appears to be mediated by cytochrome p-450 (CYP450)-generated metabolites. CYN also induces genotoxic effects both in vitro and in vivo, and preliminary evidence suggests that tumors are generated by oral exposure to CYN. To understand the role of CYP450-activated CYN metabolites on in vitro genotoxicity, this study quantified the process in primary mouse hepatocytes using the COMET assay in both the presence and absence of CYP450 inhibitors known to block acute CYN cytotoxicity. CYN was cytotoxic at concentrations above 0.1 microM (EC50 = 0.5 microM) but produced significant increases in Comet tail length, area, and tail moment at 0.05 microM and above; hence genotoxicity is unlikely to be secondary to metabolic disruption due to toxicity. The CYP450 inhibitors omeprazole (100 microM) and SKF525A (50 microM) completely inhibited the genotoxicity induced by CYN. The toxin also inhibits production of glutathione (GSH), a finding confirmed in this study. This could potentiate cytotoxicity, and by implication genotoxicity, via reduced reactive oxygen species (ROS) quenching. The lipid peroxidation marker, malondialdehyde (MDA) was quantified in CYN-treated cells, and the effect of the reduced glutathione (GSSG) reductase (GSSG-rd.) inhibitor 1,3-bis(chloroethyl)-l-nitrosourea (BCNU) on both MDA production and lactate dehydrogenase (LDH) leakage was examined. MDA levels were not elevated by CYN treatment, and block of GSH regeneration by BCNU did not affect lipid peroxidation or cytotoxicity. It therefore seems likely that CYP450-derived metabolites are responsible for both the acute cytotoxicity and genotoxicity induced by CYN.     

Effects of cholera toxin on ornithine decarboxylase activity in mouse skin

The subcutaneous injection of cholera toxin into adult mice resulted in a sustained increase in cyclic AMP levels in mouse epidermis after a lag period of about 2 hr. An increase in ornithine decarboxylase activity occurred between 7 and 10 hr, which was maintained for at least 10 hr. The increase in decarboxylase activity was localized to the area of epidermis visually affected by cholera toxin and was unaffected by hypophysectomy, suggesting a direct effect of the toxin on the epidermal cells. The subcutaneous injection of cholera toxin also led to an increase in cyclic AMP levels in newborn mouse skin. In contrast to adult mice, newborn mouse skin contained high basal activities of ornithine decarboxylase in both the epidermal and dermal fractions. The activity in both fractions was markedly decreased following cholera toxin injection. The ability of cholera toxin to induce both epidermal and dermal ornithine decarboxylase activity developed between 10 and 21 days after birth.     

In vitro toxicity and genotoxicity assessment of disinfection by‐products,organic N‐chloramines

Disinfection by‐products (DBPs) are of concern to both water industries and health authorities. Although several classes of DBPs have been studied, and there are regulated safe levels in disinfected water for some, a large portion of DBPs are not characterized, and need further investigation. Organic N‐chloramines are a group of DBPs, which can be formed during common disinfection processes such as chlorination and chloramination, but little is known in terms of their toxicological significance if consumed in drinking water. Only a few in vitro studies using bacterial assays have reported some genotoxic potential of organic N‐chloramines, largely in the context of inflammatory processes in the body rather than exposure through drinking water. In this study, we investigated 16 organic N‐chloramines produced by chlorination of model amino acids and amines. It was found that within the drinking water‐relevant micromolar concentration range, four compounds were both cytotoxic and genotoxic to mammalian cells. A small reduction of cellular GSH was also observed in the treatment with these four compounds, but not of a magnitude to account for the cytotoxicity and genotoxicity. The results presented in this study demonstrate that some organic N‐chloramines, at low concentrations that might be present in disinfected water, can be harmful to mammalian cells. © Environ. Mol. Mutagen. 2012. Published 2011 Wiley‐Liss, Inc.