ESTABLISHMENT OF THE IN VITRO DOSE-RESPONSE CALIBRATION CURVE FOR X-RAY-INDUCED MICRONUCLEI IN HUMAN LYMPHOCYTES.

The cytokinesis-block micronucleus assay has proven to be a reliable technique for biological dosimetry. This study aimed to establish the dose-response curve for X-ray-induced micronucleus. Peripheral blood samples from three healthy donors were irradiated with various doses and scoring criteria by the micronuclei (MN) in binucleated cells. The results showed that the frequency of MN increased with the elevation of radiation dose. CABAS and Dose Estimate software were used to fit the MN and dose into a linear quadratic model, and the results were compared. The linear and quadratic coefficients obtained by the two software were basically the same and were comparable with published curves of similar radiation quality and dose rates by other studies. The dose-response curve established in this study can be used as an alternative method for in vitro dose reconstruction and provides a reliable tool for biological dosimetry in accidental or occupational radiation exposures.

Genomic instability induced by ionizing radiation in human hepatocytes.

The aim of this study was to characterize genomic instability induced by ionizing radiation (IR) in human hepatocytes as reflected by alterations in cloning efficiency, micronucleus (MN) frequency, and apoptosis. The human normal liver 7702 cell line (HL7702) was subjected to initial irradiation of (60)Co-γ ray at doses of 0 (control group), 2, 4, 6, 8, or 10 Gy in each group. Progeny of surviving cells from a second irradiation at dose of 2 Gy were cultured for 15 passages until they were transferred. The cloning efficiency, MN frequency, and apoptotic rate were measured after the initial irradiation, and repeated at passage 15 before and after the second irradiation. The initial irradiation resulted in a dose-dependent decline in cloning efficiency and an increase in MN frequency and apoptotic rate. At passage 15 in progeny of initially irradiated cells, cloning efficiency, MN frequency returned to control levels while apoptotic rate rose. After the second irradiation, cloning efficiency fell while a rise in MN frequency and apoptosis occurred. Our results show that the second irradiation may further enhance cell progeny injury induced by initial irradiation, such that genomic instability that may be difficult to detect after one irradiation is more apparent with subsequent irradiation.

Proteomic alterations in progeny of irradiated human liver cells.

This study was designed to characterize the differential protein expression in the progeny of human liver cells surviving exposure to ionizing radiation. The progeny of irradiated cells were derived from a human liver cell line exposed to 0, 2, 4, or 6 Gy of (60)Co gamma-irradiation. Total protein of the cells was extracted by two-dimensional electrophoresis (2-DE) and analyzed with ImageMaster 2D Platinum software. In total, 42 differentially expressed proteins from the progeny of irradiated cells were screened, of which 17 were identified by matrix assistant laser desorption ion-top flight-mass spectrometry (MALDI-TOF-MS) analysis. There were 4 upregulated and 13 downregulated proteins detected. The upregulated expression of two proteins, mitochondrial heat-shock 60-kD protein (HSP60) and globin transcription factor 1 (GATA-1), was further confirmed by immunoblotting. Database search revealed that these differentially expressed proteins may function in cell cycle regulation, cytoskeleton maintenance, stress response, and tumor metastasis, indicating an effect of radiation-induced genomic instability (RIGI) in the progeny of irradiated cells. Analysis on functional roles of the screened proteins may provide insight into further mechanistic investigations underlying molecular events induced by RIGI.

[EoRab43 regulating vesicular transport around the macronucleus in Euplotes octocarinatus].

Rab family proteins play a crucial role in regulating vesicular traffic in eukaryotic cells. EoRab43 is an atypical Rab gene identified in Euplotes octocarinatus. In order to understand the function of EoRab43, the 153bp fragment of the 3'-end of EoRab43 gene was subcloned into expression vector pGEX-6P-1, and the recombinant plasmid pGEX-EoRab43(153bp) was transfered into E.coli BL21 (DE3) to express the fusion protein. The fusion protein GST-EoRab43C was expressed and purified by affinity chromatography. BALB/c mice were immunolized by purified GST-EoRab43C. The titer of anti-EoRab43C polyclonal antibody was detected by indirect ELISA assay and the specificity of the antibody was detected by Western blot. Immunofluorescence experiments were performed using anti-EoRab43C antibody in the cells of Euplotes. The results showed that EoRab43 displayed a punctuate pattern in the cytoplasm around the macronuclear chromosome of Euplotes.