Low-cost metaphase finder system.

In counting chromosome aberrations at low-dose radiation exposure in biological dosimetry, an automation technique has been required to process a large number of sample preparations. The metaphase finder is an automated optical microscope system, which automatically scans and finds metaphase cells on the slide glass in low magnification and relocates metaphase cells to the center of the field of view of the microscope to observe chromosomes in high magnification. The authors have constructed a cost-effective metaphase finder system by assembling commercially-available components, such as microscopes, motorized sample stages, personal computers and general-purpose image analysis software, instead of purchasing one dedicated system. The new system has high cost-effectiveness and high flexibility in adapting to the new staining methods. Mathematical morphology-based image processing to the algorithm was used to find metaphase cells. In particular, the morphology for the gray-image was used for the newest version. The performance of this metaphase finder system was tested. The new machines were distributed to six institutes of the Chromosome Network for Biodosimetry in Japan for testing the new algorithm for practical use. The scanning speed was 14.5 to 18.1 min cm2. The system was slower than several commercial metaphase finders, but there were few false positives. This system is economical and satisfactory for practical use.

Dose limits below which the effect of radiation on health becomes undetectable due to background variation.

To clarify the low-dose limit at which the effect of radiation on health becomes undetectable is important in the regulation of radiation. As one of a series of cytogenetical studies on the effect of radiation on health, we present low-dose limits determined by analyzing the background frequencies of translocations in the lymphocytes of people living in normal circumstances. The frequencies of translocations in the lymphocytes were analyzed in 20 non-smokers (61.2-year-old on the average) in a large city, and 16 non-smokers (64.4-year-old on the average) and 8 children (12.3-year-old on the average) in a remote village. The radiation dose was calculated based on the background frequencies of translocations assuming that all the translocations had been induced by radiation. The calculated doses were 384+/-200, 336+/-124 and 128+/-80 mSv in the case of chronic exposure, and 248+/-153, 225+/-104 and 107+/-72 mSv in acute exposure. Standard deviation of the calculated doses is considered to be the dose level below which the effect of radiation becomes undetectable due to the background variation in the effects of all kind of mutagenic factors, i.e., the dose level below which an epidemiological study will not be able to show any significant increase in malignant diseases. The results obtained from epidemiological studies are in fairly good agreement with our results.

Growth factor-mediated interaction between tumor cells and stromal fibroblasts in an experimental model of human small-cell lung cancer.

Malignant tumors induce development of their own stromal tissues during the processes of growth, progression and metastasis. Since the vascular architecture among the various stromal elements is well known to facilitate tumor growth and has been a target of therapy, the importance of stromal fibroblasts has recently been established. To elucidate the interaction between the tumor and its stromal fibroblasts, the present study took advantage of a unique experimental model consisting of a human small-cell lung cancer cell line, WA-ht, and its mouse stromal fibroblast cell line, WA-mFib, both originally derived from a xenograft tumor in a mouse subcutis. Co-culture with the WA-mFib cells significantly augmented the plating efficiency of WA-hT cells in vitro, and their co-inoculation in nude mice shortened latency and tumor doubling time. Histochemical detection of beta-gal, transfected into WA-mFib cells, demonstrated their contribution to the nude mouse xenograft tumor formation as its tumor stroma. Elevated hepatocyte growth factor (HGF) from fibroblasts followed by elevated production of vascular endothelial growth factor (VEGF) from both tumor cells and fibroblasts were demonstrated by ELISA in supernatants of their co-culture, accompanied by enhanced colonogenicity of the tumor cells; these enhanced features were not observed in their respective monocultures. Antisense oligonucleotides to HGF cancelled these augmentation effects with co-culture. The findings highlight the substantial roles of tumor stromal fibroblasts, interacting with soluble growth factors, in promoting the malignant propensity of the tumor.

Effect of smoking on chromosomes compared with that of radiation in the residents of a high-background radiation area in China.

Cytogenetic investigation of stable-type aberrations (translocations) was carried out with our improved methods on 28 elderly individuals in a high-background radiation area (HBRA) in China, and on 24 elderly individuals in a control area (CA). The level of radiation in HBRA is 3 to 5 times higher than in CA. The mean frequencies of translocations per 1,000 cells in HBRA and CA were 12.4 +/- 5.3 and 10.0 +/- 3.8, respectively. No significant difference was found in the frequencies between HBRA and CA (P>0.05, Mann-Whitney U test). When elderly individuals in HBRA and CA were classified into four subgroups of HBRA nonsmokers, HBRA smokers, CA nonsmokers, and CA smokers, a significant difference was found in the frequencies between CA smokers and CA nonsmokers (P<0.05, Mann-Whitney U test). Furthermore a tendency of difference (a near T-value of 0.05 level) was found in a comparison of HBRA smokers vs. CA nonsmokers. The present results indicate that the elevated level of natural radiation in HBRA plays a less significant part than smoking in bringing about the induction rate of stable-type aberrations (translocations) in those areas.

Characterization of ionizing radiation-induced ring chromosomes by atomic force microscopy.

We applied atomic force microscopy (AFM) to the structural analysis of radiation-induced ring chromosomes. Constrictions observed on the metaphase ring chromosome were found to correspond to the centromere regions of the ring chromosome in comparison with the AFM image of the centromere of rod chromosomes and with the fluorescence in situ hybridization (FISH) technique. Section analysis by AFM revealed that some ring-like chromosome fragments and ring-like chromatid fragments were thicker than standard chromosomes or chromatids, suggesting that they were ring chromosomes viewed edge on. Topographic analysis by AFM makes it possible to distinguish a ring viewed edge on that is difficult to recognize as a ring by light microscopy and to discriminate between a centric ring chromosome and an acentric ring chromosome using the same slides prepared for light microscopy.

Imperceptible effect of radiation based on stable type chromosome aberrations accumulated in the lymphocytes of residents in the high background radiation area in China.

Cytogenetic investigation of stable type aberrations (translocations) was performed with our improved methods in 6 children and 15 elderly persons in a high background radiation area (HBRA) in China, and in 8 children and 11 elderly persons in a control area. The total numbers of cells analyzed in elderly persons were 68,297 in HBRA and 35,378 in controls and in children were 45,535 in HBRA and 56,198 in controls. On average 5138 cells per subject were analyzed. The variation in the frequencies of translocations per 1000 cells was small in children while it was large in elderly persons. No significant difference was found in the frequencies between HBRA and control (P > 0.05, Mann-Whitney U test). On the other hand, correlation between age and translocation frequencies was significant at the 1% level (rs = 0.658 with 37DF, Spearman rank correlation test). The contribution of an elevated level of natural radiation in HBRA in China to the induction of stable type chromosome aberrations does not have a significant effect compared with the contribution of chemical mutagens and/or metabolic factors. The present study suggests that the probability of the risk of causing malignant and/or congenital diseases by the increased amount of radiation is imperceptible in HBRA where the level of natural radiation is 3 to 5 times higher than that in the control area.