Initial studies examined the effect of different doses of genistein given intraperitoneally (dissolved in 1.4 µmol DMSD) immediately after lower or whole-lung irradiation. DNA damage (MN assay) was assayed at 18–20 h after irradiation. These results () demonstrated that complete protection against DNA damage both in and out of field could be obtained with doses of 12.5 mg/kg and above. Preliminary studies also suggested that four doses of 50 mg/kg genistein given i.p. immediately after irradiation and then daily for the next 3 days provided some protection against increased breathing rates after both 15.5 and 18 Gy (data not shown). Based on these findings, we designed our experiment to examine the effects of putting the animals on a diet containing 750 mg/kg genistein immediately after 18 Gy to the whole lung, a dose that was chosen to achieve significant functional effects. The diet was chosen to be more appropriate for clinical application and was expected to be equivalent to a dose of approximately 20–25 mg/kg (see Methods section). Measurements in a separate group of 17 rats after 4, 8 or 14 weeks on the diet gave a mean plasma level of free genistein of 55.9 (SD 52.2) nmol/liter with no significant differences between the times.
FIG. 1 Micronucleus yields after lower (panel a) or whole (panel b) lung irradiations (10 Gy) with or without genistein treatment assayed at 18–20 h after irradiation. Panel a: Genistein was injected i.p. (50 mg/kg) 30 min prior to irradiation or 5 min (more ...)
The percentages of animals surviving after the 18 Gy whole-lung irradiation with either the control diet or genistein diet are shown in . The rats on the control diet showed a sharp decrease in survival between 40 and 80 days (approximately 6–12 weeks), with almost 50% of the rats having to be euthanized between 40 and 60 days. Rats on the genistein diet did not become moribund and were not euthanized until later, with the main effect on survival occurring between 70 and 150 days (approximately 10–21 weeks). At the end of the experiment, 28 weeks postirradiation, there was no difference in survival between the two groups.
Survival after 18 Gy whole-lung irradiation. The squares represent rats on the control (soy-free) diet and the diamonds represent rats on the genistein diet.
shows the average breathing rates for rats over the 28 weeks after irradiation. The rats irradiated with 18 Gy and put on the control diet demonstrated two waves of increase in breathing rate. The first increase occurred between 40 and 80 days (approximately 6–12 weeks) with a peak (approximately 310 bpm) at approximately 75 days postirradiation. The second increase in breathing rate occurred between 100 and 170 days postirradiation with a shallower peak (approximately 300 bpm) at 140 days. The rats on the genistein diet after irradiation also showed two waves of increase in breathing rate. However, the first increase, occurring between 40 and 70 days after irradiation, was damped compared to that seen in the control rats (peak at approximately 250 bpm at 55 days). The second wave occurring between 100 and 185 days postirradiation was similar to that for the rats on the control diet (shallow peak at approximately 310 bpm). With no irradiation, the breathing rates for rats on both the control and the genistein diet stayed at baseline levels, between approximately 180 and 220 breaths per minute, over the 28 weeks. The overall effect of genistein on breathing rate was not significant. The effect was also insignificant for the pneumonitis phase. However, the overall breathing rate of both the irradiated only and irradiated with genistein groups were significantly different from the control and control with genistein groups (P = 0.019 and P = 0.03, respectively). For the rats that survived to the end of the experiment in both irradiated groups during the pneumonitis phase, the effect was not significant.
The average breaths per minute of unirradiated rats on the control or genistein diet and of rats irradiated with 18 Gy to the whole lung and placed on the control or genistein diet. Each point represents the mean ± SEM.
The results of a quantitative analysis of DNA damage (micronuclei) in the lung cells for the animals that survived to 28 weeks are shown in . Unirradiated rats on either the control or genistein diet had background levels of DNA damage (approximately 26 micronuclei per 1000 binucleate cells). After 18 Gy whole-lung irradiation, there was a high level of DNA damage in rats on the control diet. However, rats fed the genistein diet had very low levels of DNA damage, with micronucleus formation at control (background) levels.
FIG. 4 Micronuclei per 1000 binucleate cells in lung fibroblasts. Control represents the combined groups of no radiation and control diet and no radiation and genistein diet. The radiation group was given 18 Gy whole-lung irradiation and placed on a control (more ...)
The inflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α and TGF-β were analyzed at the protein level by immunohistochemical staining in lung tissue from rats that survived to 28 weeks, as shown in . No differences were observed in the expression of any of the cytokines between rats on the genistein and control diets after sham irradiation, so the control indicates the average value of these two groups. At 28 weeks after irradiation there were significantly higher levels of IL-6 and IL-1α relative to control values, and no difference was seen between the radiation only and radiation plus genistein groups. For IL-1β, TGF-β and TNF-α, a significant increase was observed after irradiation compared to the control groups; however, when genistein was given in the diet, the irradiated group showed levels similar to those in the control rats.
FIG. 5 Protein expression of IL-1α, IL-1β, IL-6, TNF-α and TGF-β. Percentage positivity is calculated as the number of positive pixels/number of positive and negative pixels. Control represents the combined groups of no radiation (more ...)
Results of the analysis of macrophage activation at 28 weeks after 18 Gy whole-lung irradiation are shown in . No difference was seen between the genistein and control diet groups after sham irradiation, so the control represents both of these groups. After irradiation, the percentage positivity of ED-1 staining was significantly increased compared to control values. When genistein was given in the diet after irradiation, the activated macrophages levels were significantly reduced to levels similar to those seen for the control rats.
FIG. 6 Positivity of ED-1, a marker for activated macrophages in the rat. Control represents the combined groups of no radiation and control diet and no radiation and genistein diet. The radiation group was given 18 Gy whole-lung irradiation and placed on a (more ...)
Masson’s Trichrome stains collagen fibers and is therefore a marker of collagen deposition and fibrosis. Results using this stain on lung tissue from rats that survived to 28 weeks after irradiation are shown in . No difference was seen between the genistein and control diet groups after sham irradiation, so the (old) control represents both of these groups. These (old) control rats, however, showed significantly more staining than was seen in the control rats killed at the beginning of the experiment (young). The radiation-alone group demonstrated significantly more staining than the control groups. When genistein was given after irradiation, a significant decrease in positive staining was observed, although the levels remained above those seen in the control (old) rats.
FIG. 7 Masson’s Trichrome staining for collagen content (panel a) and Sircol Assay quantifying soluble collagen (panel b). Control (young) animals were assayed at approximately 8 weeks of age. Control (old) animals approximately 35 weeks of age include (more ...)
Results from the Sircol Assay are also shown in . This method can be used to quantify the amount of soluble collagen (recently synthesized collagen) in tissue. As with the Masson’s Trichrome results, the old control rats had more soluble collagen than the young control rats. After irradiation, the quantity of soluble collagen increased significantly to well above the levels in the young control rats; however, the levels were not significantly different from those of the (old) control rats that were killed 28 weeks after irradiation. After genistein administration, a decrease in the quantity of soluble collagen was observed; however, this decrease was not significant.
shows analysis of 8-oxo-deoxyguanosine (8-OHdG) staining. 8-OHdG is a specific marker for oxidative DNA damage and is used to assay for oxidative stress. As in previous figures, no difference was seen between the genistein and control diet groups after sham irradiation. No difference in the percentage positivity of staining was seen between control rats killed at the end of the experiment (old) and control animals killed at the beginning of the experiment (young). After irradiation the percentage positivity of 8-OHdG was significantly higher than that for control groups. When genistein was administered after irradiation, the amount of oxidative damage decreased significantly to levels similar to those seen in control rats.
FIG. 8 Oxidative damage in the lung assessed by the percentage positivity of 8-OHdG expression (panel a) and TBARS assay (panel b). Control (young) animals were assayed at approximately 8 weeks of age. Control (old) animals include the combined groups of no (more ...)
The concentrations of MDA are also shown in . MDA is a product of lipid peroxidation and therefore can be used as a marker of oxidative damage to membranes. The MDA concentrations were similar for the old and young controls. After irradiation the concentration of MDA increased compared to control values, though not significantly. When genistein was given in the diet after irradiation, the MDA concentrations levels were slightly higher (not significant) than those after irradiation alone.