Radiation-induced fibrosis (RIF) is a major side effect of radiotherapy in cancer patients with no effective therapeutic options. RIF involves excess deposition and aberrant remodeling of the extracellular matrix (ECM) leading to stiffness in tissues and organ failure. Development of preclinical models of RIF is crucial to elucidate the molecular mechanisms regulating fibrosis and to develop therapeutic approaches. In addition to radiation, the main molecular perpetrators of fibrotic reactions are cytokines, including transforming growth factor-β (TGF-β). We hypothesized that human oral fibroblasts would develop an in vitro fibrotic reaction in response to radiation and TGF-β. We demonstrate here that fibroblasts exposed to radiation followed by TGF-β exhibit a fibrotic phenotype with increased collagen deposition, cell proliferation, migration and invasion. In this in vitro model of RIF (RIF iv ), the early biological processes involved in fibrosis are demonstrated, along with increased levels of several molecules including collagen 1α1, collagen XIα1, integrin-α2 and cyclin D1 mRNA in irradiated cells. A clinically relevant antifibrotic agent, pentoxifylline, and a curcumin analogue both mitigated collagen deposition in irradiated fibroblast cultures. In summary, we have established an in vitro model for RIF that facilitates the elucidation of molecular mechanisms in radiation-induced fibrosis and the development of effective therapeutic approaches.

An adaptive response has been demonstrated in certain mammalian cells wherein pre-exposure to a small radiation dose prior to a large dose ameliorates the damage induced by the second dose. We investigated whether a similar response could occur in the developing brain of the fetal rat, and if so, what the optimum interval between the two doses would be. Pregnant rats were exposed to a dose of 0.02 Gy γ radiation at variable times (1, 3, 6, 12, or 24 h) prior to a second dose of 0.5 Gy on day 15 of gestation. Fetuses were harvested at 6 and 24 h after the second irradiation and standard cellular morphological assessments performed on the developing cerebral cortex. For number of mitotic cells, pyknotic cells, and macrophages, no significant differences were found between any of the groups that had received the priming (0.02 Gy) dose and the group that had not. Significant differences were found between fetuses harvested at 6 h and those harvested at 24 h after the final irradiation for all parameters measured. Thus, while the data were consistent with past research relating to the effects of radiation on the development of the brain of the fetal rat, no evidence for an adaptive response to radiation was found. Whether an adaptive response was indeed absent, or whether the doses and/or intervals used were simply not appropriate for demonstrating it, remains unknown.

Therapeutic thoracic irradiation may induce two late pulmonary injury syndromes: radiation pneumonitis and subsequent pulmonary fibrosis. The alveolar macrophage has been considered a radioresistant cell and not a target cell involved in the pathogenesis of either type of radiation-induced lung injury. Alveolar macrophage-derived cytokines, including interleukin-1 (IL-1) and tumor necrosis factor (TNF), have been demonstrated to participate in inflammatory and fibrotic responses in the lung after various other types of lung injury. To evaluate whether the release of cytokines by alveolar macrophages is induced by radiation doses used clinically, alveolar macrophages recovered from nonsmoking volunteers were exposed in vitro to a single dose of 2 Gy and then maintained in culture for 18 h. Culture supernatants and cell lysates were then recovered and analyzed for IL-1α and IL-1β by radioimmunoassay. Supernatants of irradiated alveolar macrophages contained significantly increased amounts of IL-1α (P < 0.04) and IL-1β (P < 0.02) as well as total IL-1 (IL-1α and IL-1β) (P < 0.02) compared to nonirradiated alveolar macrophages. Cell lysates of irradiated alveolar macrophages also contained increased amounts of IL-1α and IL-1β, although differences from controls were not significant. The finding of increased release of IL-1 by alveolar macrophages after exposure to a single, clinically relevant dose of radiation suggests that the function of human alveolar macrophages is likely altered during therapeutic use of thoracic irradiation. Whether this release of IL-1 by alveolar macrophages contributes to early lung inflammation induced by thoracic irradiation is unclear.

Based on previous studies showing that exposure of the rat fetus to ionizing radiation produces dose-dependent (0.25-1.25 Gy) changes in postnatal development of behavior and decreases in the thickness of the cerebral cortex, we examined the extent to which dose fractionation would reduce expression of damage. Pregnant rats were exposed to single doses of 0.5 or 1.0 Gy, or to two doses of 0.5 Gy 6 h apart on day 15 of gestation. Offspring were subjected to four behavioral tests on postnatal days 7-28; the rats were then sacrificed and their brains removed and processed for histology. For all end points, the fractionated dose produced an effect that was intermediate between that of the 0.5- and 1.0-Gy doses and which, by interpolation, could be expressed as equivalent to a single dose between 0.5 and 1.0 Gy. The equivalent single dose was not significantly different from the 1.0-Gy dose for negative geotaxis (0.54 Gy), reflex suspension (0.80 Gy), and continuous corridor (0.85 Gy). For sine of the angle of the advance of hind feet (0.58 Gy), width of stride (0.69 Gy), length of stride (0.75 Gy), body weight (0.73 Gy), and cerebral cortex thickness (0.69 Gy), the fractionated dose produced effects significantly different (P < 0.05) from the 1.0-Gy dose. Overall, exposure of fetal rats to two doses of 0.5 Gy separated by 6 h produced effects equivalent to a single dose of 0.70 Gy, as measured by postnatal behavioral tests and morphological assessment of brain structure.

Pregnant rats were exposed to γ radiation from a 137 Cs irradiator on gestational Day 15. Fetuses that received 0.25, 0.5, 0.75, or 1.0 Gy were examined 24 h after irradiation for changes in the cells of the cerebral mantle of the developing brain. The extent of changes following 0.5 Gy was studied at 3, 6, 12, or 24 h after exposure. Cortical thickness of the cerebral mantle was not significantly altered. The number of pyknotic cells, number of macrophages, nuclear area, and number of mitotic cells were altered in a dose-related way. The number of pyknotic cells was significantly increased at all doses. A positive correlation between the number of pyknotic cells and the number of macrophages developed with time. At 3 h after irradiation about 60% of pyknotic cells were found in the subventricular zone and about 25% in the intermediate zone and cortical plate. The number of such cells in the upper layers of the cortex steadily increased up to 24 h, at which time about 70% of pyknotic cells were in these two layers. The relationship of the movement of pyknotic cells to migration of postmitotic neuroblasts is discussed.

The purpose of this study was to quantitate cell populations recovered by lung lavage up to 6 weeks following thoracic irradiation (24 Gy) as an index of the acute inflammatory response within lung structures. Additionally, rats were treated five times weekly with intraperitoneal saline (0.3 cc) or methylprednisolone (7.5 mg/kg/week). Lung lavage of irradiated rats recovered increased numbers of total cells compared to controls beginning 3 weeks after irradiation (P < 0.05). The initial increase in number of cells recovered was attributable to an influx of neutrophils (P < 0.05), and further increases at 4 and 6 weeks were associated with increased numbers of recovered macrophages (P < 0.05). Lung lavage of steroid-treated rats at 6 weeks after irradiation recovered increased numbers of all cell populations compared to controls (P < 0.05); however, numbers of recovered total cells, macrophages, neutrophils, and lymphocytes were all significantly decreased compared to saline-treated rats (P < 0.05). The number of inflammatory cells recovered by lung lavage during acute radiation-induced lung injury is significantly diminished by corticosteroid treatment. Changes in cells recovered by lung lavage can also be correlated with alteration in body weight and respiration rate subsequent to treatment with thoracic irradiation and/or corticosteroids.

Dihydroxyanthraquinone (DHAQ) is currently being tested as a cancer chemotherapeutic agent because of its structural similarity to Adriamycin (ADR) and other DNA-intercalating antibiotics. The interaction of DHAQ and ionizing radiation on the induction of cell lethality was investigated in Chinese hamster ovary cells in culture. In asynchronous populations of cells, DHAQ produced a slight enhancement of radiation-induced cell lethality as evidenced by changes in both shoulder and slope of the radiation dose-survival curves. However, DHAQ had no effect on either the extent or time course of recovery from sublethal radiation damage. In synchronous populations of cells treated at various times before or after selection in mitosis, the combination of DHAQ and radiation produced greater cell killing than that predicted based on simple additivity of effect, with a decided enhancement for cells treated during S phase. These results indicate that DHAQ is similar to other DNA-intercalating antibiotics in regard to the interaction with ionizing radiation to produce cell lethality.

The response of cultured 9L rat brain tumor cells to hyperthermia (45°C water bath) and ionizing radiation was investigated as a function of their position in the cell cycle. Similar to other cell lines, 9L cells demonstrated a sensitivity to cell killing by hyperthermia during S phase, and in particular late S phase, in contrast to high survival in early G 1 phase. Mitotic cells treated prior to selection under proper pH and temperature conditions displayed a survival that was comparable to that of S-phase cells. Using the method of retroactive synchrony, the thermal sensitivity during mitosis was also shown to fluctuate, being greater for cells closer to division than those near the G 2 / M boundary. Irradiated 9L cells exhibit only minimal changes in survival across the majority of the cell cycle (except for a radiosensitive mitosis). Combining the two agents resulted in survival levels that were less than expected from the product of the survivals after individual treatments. The degree of enhancement was greatest for those cells in mid- G 1 phase and S phase. When the two treatments were separated in time, there was an increase in survival that was greater when X rays preceded heat than for the reverse sequence. The degree of recovery was greater for cells initially treated at the S/ G 2 boundary, than at the G 1 / S boundary, than at mid- G 1 . Recovery to an independent level was reached within 1 hr for all positions except mid- G 1 when radiation preceded hyperthermia; for the reverse sequence, recovery to an independent level was not observed over a 4-hr period.

Several responses of cultured 9L rat brain tumor cells to ionizing radiation were investigated as a function of their position in the cell cycle. The mitotic selection procedure for cell cycle analysis was utilized to study the blockade of progression of cells through G 2 and the resultant division delay. The transition point for this blockade (i.e., the last point in the cell cycle at which cells are blocked, and after which they are refractory to delay) was located approximately at the G 2 / M boundary 35 min prior to selection. The duration of division delay was a linear function of the X-ray dose, 33 min of delay/Gy. The survival of cells following exposure to a constant dose of X rays at various times after incubation of mitotically selected cells demonstrated a definite resistance during the G 1 phase and slight resistance during the S phase, relative to the level of survival at the G 1 / S boundary. As shown with other cell lines there was maximum radiosensitivity during G 2 and mitosis. Thus the response of 9L rat brain tumor cells in culture to ionizing radiation is similar to that of other cultured mammalian cell lines in regard to induction and duration of division delay, and the sensitivity of M and G 2 cells to radiation-induced cell lethality. However, the radioresistance exhibited over the remainder of the cell cycle results in a relatively flat age response for radiation-induced lethality. This may in part explain the radiation resistance observed for the 9L gliosarcoma in situ.

The mitotic selection procedure for cell cycle analysis was utilized to investigate the G 2 transition point for and the duration of radiation-induced division delay in diploid and tetraploid Chinese hamster ovary (CHO) fibroblasts and in Chinese hamster ovarian carcinoma cells. The location of the radiation-induced division delay transition point, i.e., the last point in the cell cycle at which X irradiation can block progression, was dose independent at high doses (approximately 1.5 Gy and above) and located approximately 42 min before division. At lower doses, as more cells were refractory to delay, only an estimate of the point of blockade was possible; but the G 2 transition point appeared to be earlier in the cell cycle, e.g., 54 min at 0.25 Gy. The duration of radiation-induced division delay was dose dependent. At low doses there was a nonlinear rapid increase in delay up to approximately 1.0 Gy, and from 1.0 to 6.0 Gy there was a linear dose response of approximately 50 min/Gy as determined from over 80 experiments conducted over 3 years. This response is consistent with a sensitive population of cells in late G 2 that define the location of the transition point and the length of division delay. There was no difference observed in the dose response for radiation-induced division delay between the pseudotetraploid cell line of CHO (<tex-math>$T_{{\rm D}}=13$</tex-math> hr, N̄ = 40 chromosomes, delay = 61 ± 2 min/Gy) and the pseudodiploid parent strain (<tex-math>$T_{{\rm D}}=13$</tex-math> hr, N̄ = 21 chromosomes, delay = 62 ± 2 min/Gy). However, in the cell line derived from a spontaneous Chinese hamster ovarian carcinoma (<tex-math>$T_{{\rm D}}=11.5$</tex-math> hr, N̄ = 40 chromosomes) the division delay was 39 ± 4 min/Gy. Therefore, radiation-induced division delay is independent of chromosome ploidy, but can show intraspecies cell line specificity.

The presence of 0.75 μM N-ethylmaleimide (NEM) during irradiation of synchronous HeLa cells at different stages of their cycle enhances cell killing (i.e., sensitizes) in early G 1 and late S, but does not affect mitotic cells or cells at the G 1 / S border. The effect is primarily on the shoulder of the survival curve, rather than its slope. NEM given immediately before or after irradiation is as effective as during irradiation. When given as a function of time after exposure the effect decreases in magnitude, cells becoming insensitive to NEM more rapidly in S than in G 1 . The drug is also effective if administered prior to irradiation, the effect generally decreasing the longer the interval between treatment and irradiation. The addition of 1.0 mM hydroxyurea (HU) to synchronous HeLa cells in G 1 showed that survival changes occur as the cells proceed through the "cell cycle" even in the absence of DNA synthesis. The changes observed are qualitatively the same to those observed previously in Chinese hamster cells. The irradiation of HU-inhibited cells in the presence of 0.75 μM NEM produced almost no variation in survival through "the cycle," the inhibited cells showing a sensitivity greater than that normally observed in mitotic cells. The results presented are consistent with the idea that NEM inhibits repair processes in the cell most likely due to its binding to a critical SH-containing enzyme(s) concerned with the repair of radiation damage.