Imaging and microanalysis of 14N and 15N by SIMS microscopy in yeast and plant samples.

We have investigated the usefulness of Secondary Ion Mass Spectrometry (SIMS) for studying the tissue distribution of 15N labelling in yeast cells and soybean leaf tissues. The secondary ions best suited for this are 12C14N- and 12C15N-. Using a mass resolution of 6000, all problems of interference by other ions were avoided. The lateral resolution was of the order of 300 nm, i.e. well suited for subcellular studies. The sensitivity was good enough to allow the detection and the mapping of 15N, even when it was present at its natural value concentration of the isotopic ratio of only 0.37%. Using yeast cells at isotopic equilibrium with their nutrient medium, the nitrogen isotopic ratios in the cells were consistent with those in the medium. In the soybean leaf samples, the mapping of 14N and 15N was well correlated with the anatomical structures of the tissues. The mean isotopic ratios (100 15N/14N, at/at), measured in the leaf tissues by SIMS, were slightly below those in the nutrient medium as well as those measured in the leaf tissue by conventional mass spectrometry. This may be explained by differences in the methods of preparation of the leaf samples for SIMS and for mass spectrometry, and by the fact that the plants were probably still not perfectly at isotopic equilibrium with their external medium at the time the experiments were performed.

The (8-thiotheophyllinate) (triphenylphosphine) gold (I), (tTAuP): a new gold complex as an anticancer agent.

In continuous Friend leukemia cell exposure to tTAuP, the IC50 was 0.2 microM whereas in cells exposed 15 or 60 min to tTAuP followed by 72 h in drug-free medium the IC50 was 2.2 and 1.3 microM respectively. A combination of tTAuP and cisplatin (CDDP) is shown to be more active than either agent alone. Intracellular accumulation studies analysed by SXRF have shown that to achieve a cytotoxic effect, large concentrations of gold are necessary to accumulate in both the nuclear and cytoplasmic fraction. However, at cytostatic doses (1 microM), tTAuP has no effect on the cell cycle but does affect DNA synthesis. At a higher dose, greater than that necessary to induce cytotoxicity, it produces DNA damage, as observed by alkaline elution method. When cells were exposed to toxic doses of tTAuP (10 microM) in the presence of albumin, cytotoxicity was significantly reduced. Similar results were obtained when cells were co-treated with L-cysteine, dithiothreitol or reduced gluthatione. Reduced cytotoxic effect can be related to the interaction with free thiol groups. According to these data it is concluded that tTAuP is highly effective in vitro; whether it is active in vivo remains to be determined.

Intracellular accumulation and cytotoxic effect of (8-thiotheophyllinate) (triphenylphosphine) gold(I) in Friend leukemia cells.

Following continuous exposure to tTAuP in medium containing 10% fetal calf serum (FCS), cells resistant to doxorubicin (DOX-RFLC) were 3 fold more resistant to tTAuP than its sensitive counterpart (FLC). Moreover, FLC were 100 fold more sensitive to tTAuP when the cells were grown in the absence of FCS but in the presence of synthetic medium (BMS). When the cytotoxic effect was measured after short-term exposure (60 min) followed by 72 hr incubation in drug-free medium, unexpectedly, DOX-RFLC were 8 fold more sensitive [ID50 = 1.5 microM] than FLC [ID50 = 12 microM] to tTAuP. When FLC were exposed 60 min at different temperatures (4, 25 or 37 degrees C) to tTAuP prior to 72 hr incubation in drug free medium, the ID50 was achieved at 20, 13 and 3 microM respectively). In contrast, when DOX-RFLC were treated in similar conditions, the cytotoxic activity of tTAuP did not vary following exposure at 4, 25 or 37 degrees C. Although the mechanism of action of gold compounds is still unknown we have assumed that tTAuP cytotoxicity is related to the ease with which it is accumulated. This assumption was supported by the analysis of tTAuP incorporation by SXRF. The results reported here show that tTAuP accumulates rapidly in the cell and is distributed in the cytoplasmic and the nuclear fractions. It is suggested that accumulation is mediated by passive diffusion. However, the detection of gold binding to DNA in complexes resistant to solvent treatments suggests that interaction with macromolecules can be mediated by the formation of covalently bound complexes.