Protective effects of melatonin against low- and high-LET irradiation

To investigate the protective effects of melatonin against high-LET ionizing radiation, V79 Chinese hamster cells were irradiated with 100 keV/mu m carbon beam. Parallel experiments were performed with 200 kV X-rays. To avoid the impact from extra solvents, melatonin was dissolved directly in culture medium. Cells were cultured in melatonin medium for 1 hr before irradiation. Cell inactivation was measured with conventional colony forming assay, medium containing 6-thioguanine was used for the selection of mutants at hprt locus, and the cell cycle was monitored by flow cytometry. Both carbon beam and X-rays induced cell inactivation, hprt gene mutation and cell cycle G2 block dose-dependently. But carbon beam showed stronger effects as indicated by all three endpoints and the relative biological effectiveness (RBE) was 3.5 for cell killing (at 10% survival level) and 2.9 for mutation induction (at 5 x 10(-5) mutants/ cell level). Melatonin showed protective effects against ionizing radiation in a dose-dependent manner. In terms of cell killing, melatonin only increased the survival level of those samples exposed to 8Gy or larger of X-rays or 6 Gy or larger of carbon beam. In the induction of hprt mutation and G2 block, melatonin reduced such effects induced by carbon beam but not by X-rays. The results suggest that melatonin reduces the direct interaction of particles with cells rather than an indirect interaction. Further studies are required to disclose the underlying mechanisms.

Deletion of yeast CWP genes enhances cell permeability to genotoxic agents

We have previously reported the development of a novel genotoxic testing system based on the transcriptional response of the yeast RNR3-lacZ reporter gene to DNA damage. This system appears to be more sensitive than other similar tests in microorganisms, and is comparable with the Ames test. In an effort to further enhance detection sensitivity, we examined the effects of altering major cell wall components on cell permeability and subsequent RNR3-lacZ sensitivity to genotoxic agents. Although inactivation of single CWP genes encoding cell wall mannoproteins had little effect, the simultaneous inactivation of both CWP1 and CWP2 had profound effects on the cell wall structure and permeability. Consequently, the RNR3-lacZ detection sensitivity is markedly enhanced, especially to high molecular weight compounds such as 4-nitroquinoline-N-oxide (> sevenfold) and phleomycin (> 13-fold). In contrast, deletion of genes encoding representative membrane components or membrane transporters had minor effects on cell permeability. We conclude that the yeast cell wall mannoproteins constitute the major barrier to environmental genotoxic agents and that their removal will significantly enhance the sensitivity of RNR-lacZ as well as other yeast-based genotoxic tests.

Nuclear transfer in leach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the leach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 degreesC in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days, It was found that gastrula cells incubated at 4 degreesC had the same totipotence as blastula cells, The optimal UV dosage for inactivation of the oocyte chromatin was 180-240 mJ cm(-2). Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.