Synthesis and biological evaluation of hybrid acridine-HSP90 ligand conjugates as telomerase inhibitors.

The synthesis and biological evaluation of a series of bifunctional acridine-HSP90 inhibitor ligands as telomerase inhibitors is herein described. Four hybrid acridine-HSP90 inhibitor conjugates were prepared using a click-chemistry approach, and subsequently shown to display comparable results to the established telomerase inhibitor BRACO-19 in the TRAP-LIG telomerase assay. The conjugates also demonstrated significant cyctotoxity against a number of cancer cell lines, in the sub-µM range.

Pretreatment of rats with the inducing agents phenobarbitone and 3-methylcholanthrene ameliorates the toxicity of chromium (VI) in hepatocytes.

To exert cytotoxicity chromium VI (Cr(VI)) has to be reduced inside cells. This is achieved through both enzymatic and non-enzymatic mechanisms. Enzymatic mechanisms include DT-diaphorase, cytochrome P450, and NADPH cytochrome c reductase, and non-enzymatic mechanisms involve reduced glutathione (GSH) and ascorbic acid. The extent of cytotoxicity of Cr(VI) may thus be influenced by the availability of non-enzymatic reductants, and by the activities of the reductase enzymes. In the present paper we have investigated the effect of pretreatment with the inducing agents, phenobarbitone (PB) and 3-methylcholanthrene (3-MC), on the response of rat hepatocytes to Cr(VI). Pretreatment with PB increased the activity of NADPH cytochrome c reductase, and 3-MC increased DT-diaphorase activity in hepatocytes. Both inducers increased cytochrome P450 content, while neither influenced intracellular GSH content or the activity of glutathione reductase. Pretreatment with either PB or 3-MC resulted in amelioration of Cr(VI) toxicity both in terms of hepatocyte viability, and to a greater extent, in terms of Cr(VI) induced GSH loss. We propose that the inducing agents increase the amount of enzymatic reduction of Cr(VI) relative to non-enzymatic reduction. Thus, less GSH is used in the reduction of Cr(VI), and intracellular GSH does not fall as rapidly as in cells from control animals therefore cell integrity is better maintained. Exposure to environmental inducing agents in vivo may also alter the response of human tissues to Cr(VI).

Chromium(VI)-induced damage to the cytoskeleton and cell death in isolated hepatocytes.

Cr(VI) is a known human carcinogen. Although it has been investigated widely, the mechanism(s) of its action is/are not fully understood. The aim of this study was to evaluate Cr(VI)-induced damage to the cell cytoskeleton and the mode of cell death in primary cultures of hepatocytes. Exposure of the cultured cells (10(5)/cm(2)) to 1 and 5 microM Cr(VI) for 24 h resulted in loss of the cell cytoskeleton, and this was accompanied by membrane blebbing and shrinking of the cell. Staining of the cells with annexin V and propidium iodide showed that Cr(VI) induces apoptosis at low concentrations (5 microM), whereas at higher concentrations (25 microM) it induces necrosis. This study shows that Cr(VI) causes damage to the cell cytoskeleton, and induces apoptosis at low concentrations. However, the importance of necrosis and apoptosis in vivo, and the effects of longer exposure times, which simulate environmental and occupational exposure to Cr(VI), remain to be investigated.

The role of glutathione reductase in the cytotoxicity of chromium (VI) in isolated rat hepatocytes.

Chromium (VI) is an environmental and occupational carcinogen, and it is accepted that intracellular reduction is necessary for DNA damage and cytotoxicity. We have investigated the interaction of Cr(VI) with hepatocytes in vitro to determine the contribution of various hepatic enzymes to the reduction of Cr(VI). Cr(VI) caused a dose-dependent decrease in cell viability and intracellular reduced glutathione (GSH) levels between 100 and 500 microM within 3 h exposure of hepatocytes. Both DT-diaphorase and cytochrome P450 play only a minor role in detoxifying Cr(VI) and/or its metabolites. (GSH) appears to act as a non-enzymatic reductant, reducing Cr(VI) to a toxic form. The evidence for this is two-fold. Firstly, GSH was depleted during the metabolism of Cr(VI) and, secondly, pretreatment of the cells with diethylmaleate to deplete GSH levels, partially protected the cells from Cr(VI) toxicity. Glutathione reductase appears to play an important role in the enzymatic reduction of Cr(VI) as inhibition of this enzyme by carmustine (BCNU) markedly protected the cells from cytotoxicity.

The interaction of the orthopaedic metals, chromium VI and nickel, with hepatocytes.

High levels of metal ions, such as chromium and nickel, released from metallic total hip implants have been detected in the serum and urine of patients. Cr VI and Ni ions are carcinogenic and toxic and there is concern about their systemic toxicity. To investigate this we have studied the interaction of Cr VI and Ni with hepatocytes. Both metal ions caused loss of cell viability within 3 h exposure, Cr VI was more potent than Ni. Cr VI caused depletion of intracellular reduced glutathione (GSH) levels, and inhibition of glutathione reductase and glutathione-S-transferase (GST) activities. Expression of alpha-GST, the major isoenzyme of GST in rat liver, was also decreased by Cr VI. Ni, on the other hand did not deplete GSH, or inhibit any of the enzyme activities measured in the cells. GSH and GST form a major protection and detoxification system in the liver, and depletion of GSH and inhibition of GST activity by Cr VI in vivo may severely compromise the ability of an individual to protect himself against carcinogenic and cytotoxic chemicals in the environment.

Building the Hospital Without Walls--a CSIRO home telecare initiative.

Aging populations and rising health costs have created the need to care for more patients in their own homes. Australia's Commonwealth Scientific and Industrial Research Organization (CSIRO) is developing a project, Hospital Without Walls, which aims to provide continuous monitoring of patients in certain diagnostic categories. The key technology is a miniature, wearable, low-power radio that can transmit vital sign and activity information to a home computer, from which data may be sent by telephone line and the Internet to appropriate medical professionals. The initial clinical scenario for this work is monitoring of elderly patients who have presented to hospitals following repeated falls. Accelerometers built into the radio sets will monitor activity and detect and characterise falls. Simultaneous measurement of heart rate will provide information about abnormalities of cardiovascular physiology at the time of a fall. The system has been tested in laboratory conditions and is being adapted for initial clinical trials.

Enumeration of subgingival species on primary isolation plates using colony lifts.

This study evaluated the feasibility of using a colony lift method and DNA probes to enumerate bacterial species cultured on primary isolation plates. Fourteen digoxigenin-labeled whole chromosomal DNA probes representing 12 subgingival species were validated by hybridization with colony lifts prepared from 249 reference strains of 51 species grown on Trypticase soy agar plates supplemented with 5% sheep blood. Colonies of reference strains were lifted onto Nytran filters from plates and treated to lyse cells, remove cellular proteins, denature and fix microbial DNA to the filters. Positive reactions were detected with an anti-digoxigenin antibody conjugated to alkaline phosphatase and revealed by bromo-chloro-indolyl phosphate and nitroblue tetrazolium. Cross-reactions were not observed for 13/14 probes, but 2 strains of Streptococcus mitis reacted with the probe to Streptococcus sanguis II. Subgingival plaque samples were taken by means of a sterile curette from mesiobuccal surfaces of teeth present in each of 26 subjects with differing periodontal disease states. Samples were dispersed, diluted, plated and incubated anaerobically for 7 d at 35 degrees C. Colonies were lifted as described above. Filters were cut into sections and hybridized with the 14 digoxigenin-labeled DNA probes. The probes were used to enumerate the test species and the total number of isolates was determined in 711 plaque samples. The colony lift method and DNA probes provided a sensitive, economical and quantitative method for enumerating cultivable microbial species in subgingival plaque samples. In addition, the amplification provided by growing the organisms on agar plates facilitated determination of numbers of organisms in small plaque samples, such as those from healthy sites.