Evidence has accumulated showing that ionizing radiations persistently perturb genomic stability and induce delayed reproductive death in the progeny of survivors; however, the linear energy transfer (LET) dependence of these inductions has not been fully characterized. We have investigated the cell killing effectiveness of gamma rays (0.2 keV/microm) and six different beams of heavy-ion particles with LETs ranging from 16.2 to 1610 keV/microm in normal human fibroblasts. First, irradiated confluent density-inhibited cultures were plated for primary colony formation, revealing that the relative biological effectiveness (RBE) based on the primary 10% survival dose peaked at 108 keV/microm and that the inactivation cross section increased proportionally up to 437 keV/microm. Second, cells harvested from primary colonies were plated for secondary colony formation, showing that delayed reproductive death occurred in a dose-dependent fashion. While the RBE based on the secondary 80% survival dose peaked at 108 keV/microm, very little difference in LET was observed in the RBE based on secondary survival at the primary 10% survival dose. Our present results indicate that delayed reproductive death arising only during secondary colony formation is independent of LET and is more likely to be dependent on initial damages having been fixed during primary colony formation.

It is thought that heavy-ion radiation therapy is a promising modality of cancer therapy that can deliver higher biological effects on tumors than conventional X-ray radiation therapy, and may also conserve the function of normal organs because of its excellent dose distribution.(1) Recently, excellent clinical results were reported in the treatment of lung cancer, prostate cancer, cancer of the head and neck, and malignant tumors of bone and soft tissue.(2) Heavy-ion has high linear energy transfer (LET) with increased relative biological effectiveness (RBE). However, the possible mechanism of increasing cell killing in heavyion therapy is not fully understood. Previous studies have demonstrated that the induction of cell killing of non-irradiated cells by adjacent cells that are directly irradiated by heavy-ions is considered to play an important role in radiation-induced cell death, not only in tumors but also in normal organs. This phenomenon is termed the "bystander effect", and was reported by Nagasawa and Little in 1992.(3) They reported that increased sister chromatid exchanges were observed in more than 30% of cells when less than 1% of the nuclei of cultured cells were actually traversed by an α-particle in Chinese hamster ovary cells. It has been proposed that the mediating mechanism involves secreted soluble factors, (4)(5)(6) an increase in oxidative stress,plasma membrane-bound lipid rafts, (8) and gap junctional intracellular communication (GJIC). (4,7,(9)(10)(11)(12)(13) Gap junctions are membrane channels that connect the cytoplasm of cells and allow direct exchange of small molecules and ions between adjacent cells. GJIC is essential for the maintenance of tissue homeostasis and proliferation and the regulation of embryonic development and differentiation, and it plays a key role in induction of the bystander effect. (14)(15)(16)(17) Here, bystander killing of cells by heavy-ion irradiation was investigated, focusing on the involvement of GJIC on cell survival, to clarify the underlying mechanisms of the bystander effect in a human lung cancer cell line in vitro. Materials and MethodsCell cultures. A549 (a human lung cancer cell line with wild-type p53) was obtained from the Cell Resource Center for Biomedical Research at the Institute of Development, Aging and Cancer, Tohoku University, Japan. Cells were cultured in RPMI-1640 medium (Immuno-Biological Laboratories, Takasaki, Japan) supplemented with 10% fetal calf serum (PAA Laboratories, Pasching, Austria). Two days prior to irradiation experiments, 5 × 10 5 and 5 × 10 4 cells were inoculated into a specially designed 35-mm microbeam dish that was constructed as previously described, (18) to prepare confluent cultures and sparsely populated cultures, respectively. Unless otherwise described, all cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO 2 in air. The size of the cell nucleus and the whole cells was determined to be 14.6 ± 4.3 and 106 ± 24.9 μm 2 , respectively. Drug treatment. Prior to irradiation, cells were p...

These results indicate that the microbeam irradiation is useful in characterizing tissue-specific, local biological response to radiation in organisms. DNA damage-induced cell cycle arrest and apoptosis were observed in locally irradiated regions, but there was little, if any, 'bystander effect' in the nematode.

Research concerning cellular responses to low dose irradiation, radiation-induced bystander effects, and the biological track structure of charged particles has recently received particular attention in the field of radiation biology. Target irradiation employing a microbeam represents a useful means of advancing this research by obviating some of the disadvantages associated with the conventional irradiation strategies. The heavy-ion microbeam system at JAEA-Takasaki, which was planned in 1987 and started in the early 1990's, can provide target irradiation of heavy charged particles to biological material at atmospheric pressure using a minimum beam size 5 mum in diameter. A variety of biological material has been irradiated using this microbeam system including cultured mammalian and higher plant cells, isolated fibers of mouse skeletal muscle, silkworm (Bombyx mori) embryos and larvae, Arabidopsis thaliana roots, and the nematode Caenorhabditis elegans. The system can be applied to the investigation of mechanisms within biological organisms not only in the context of radiation biology, but also in the fields of general biology such as physiology, developmental biology and neurobiology, and should help to establish and contribute to the field of "microbeam biology".

Brown adipose tissue (BAT) plays an important role in thermoregulation in species living in cold environments, given heat can be generated from its chemical energy reserves. Here we investigate the existence of BAT in blubber in four species of delphinoid cetacean, the Pacific white-sided and bottlenose dolphins, Lagenorhynchus obliquidens and Tursiops truncates, and Dall’s and harbour porpoises, Phocoenoides dalli and Phocoena phocoena. Histology revealed adipocytes with small unilocular fat droplets and a large eosinophilic cytoplasm intermingled with connective tissue in the innermost layers of blubber. Chemistry revealed a brown adipocyte-specific mitochondrial protein, uncoupling protein 1 (UCP1), within these same adipocytes, but not those distributed elsewhere throughout the blubber. Western blot analysis of extracts from the inner blubber layer confirmed that the immunohistochemical positive reaction was specific to UCP1 and that this adipose tissue was BAT. To better understand the distribution of BAT throughout the entire cetacean body, cadavers were subjected to computed tomography (CT) scanning. Resulting imagery, coupled with histological corroboration of fine tissue structure, revealed adipocytes intermingled with connective tissue in the lowest layer of blubber were distributed within a thin, highly dense layer that extended the length of the body, with the exception of the rostrum, fin and fluke regions. As such, we describe BAT effectively enveloping the cetacean body. Our results suggest that delphinoid blubber could serve a role additional to those frequently attributed to it: simple insulation blanket, energy storage, hydrodynamic streamlining or contributor to positive buoyancy. We believe delphinoid BAT might also function like an electric blanket, enabling animals to frequent waters cooler than blubber as an insulator alone might otherwise allow an animal to withstand, or allow animals to maintain body temperature in cool waters during sustained periods of physical inactivity.