Radiation exposure can generate a cascade of molecular and cellular events in a variety of cell types in the lungs that lead to the activation of pro-inflammatory and pro-fibrotic processes. The level of damage to the lung has been shown to be dependent on total radiation dose, whether the dose was delivered in single or multiple fractions, the locality and amount of irradiated tissue, and whether the dose was received externally or internally (radionuclides).3
Here, we investigated the ability of genistein to protect against acute radiation injury and delayed radiation-induced damage to the lung in a total body irradiation model. The results of this study show that genistein, given as a single SC injection of 200 mg/kg body weight 24 h before irradiation, significantly reduced acute radiation-induced mortality and radiation-induced weight loss in C57BL/6J female mice. This demonstrates that the radioprotection by genistein against ARS previously observed with CD2F1 male mice is neither strain- nor gender-dependent.22,23
Although we did not observe classic radiation-induced pneumonitis or fibrosis in our TBI model, we did observe some injuries in the lung including DNA damage and transient alterations in protein expression. Our investigation of genistein protection against lung injury shows that genistein reduced radiation-induced DNA damage. The genotoxicity protection correlated with genistein protection against radiation-induced collagen deposition and some other alterations in protein expression in the lung.
The CBMN assay revealed that mice receiving TBI developed micronuclei in pulmonary fibroblasts, and that genistein reduced this radiation-induced DNA damage. The micronucleus index is a standard test used in genetic toxicology to assess chromosome damage.35
This assay is the preferred method for measuring micronuclei in cultured mammalian cells because scoring is restricted to once-divided cells, eliminating cell division kinetics which can influence micronuclei frequency. We observed an eight-fold increase in the incidence of micronuclei in ex vivo
pulmonary fibroblast cultures from mice exposed to 7.75 Gy TBI, indicating the presence of radiation-induced DNA damage. Khan et al.
reported a higher incidence of micronuclei in primary ex vivo
fibroblast cultures obtained from rats 16–18 h after partial volume lung irradiation with 10 Gy 60
Co compared to fibroblasts from unirradiated lungs. The relationship between radiation-induced micronuclei and lung fibrosis, however, is not clear. Our studies showed that fibroblasts from mice treated with genistein prior to irradiation had decreased micronuclei frequency, demonstrating genistein protection against radiation-induced genotoxicity in pulmonary fibroblasts.
Histological analyses of the lung tissue from irradiated and control animals at 90, 120, and 180 days did not identify pneumonitis or classic fibrotic remodeling following 7.75 Gy (60
Co, 0.6 Gy/min) whole body irradiation. It is possible that pneumonitis occurred at earlier time points that were not examined in our experiments. Van der Meeren et al.
(2003) examined the lung from days 10–18 following 10 Gy total body irradiation and found increased expression of genes involved in pro-inflammatory and thrombotic processes.28
Mattos et al.
(2002) reported that C57BL mice exposure to 7 Gy TBI (60
Co, 0.97 Gy/min) resulted in significant collagen deposition with vascular congestion and alveolar septal thickening deposition within 30 days, and significant collagen deposition after 90 days.27,37
Although we were unable to observe classic histochemical evidence of pneumonitis or fibrosis, we did identify small, focal collagen-rich deposits or plaques in the lungs at 90 days postirradiation in animals receiving vehicle or no treatment. These lesions were not present in irradiated mice pretreated with genistein. The differences observed in our study may be the result of our lower radiation dose rate, differences in the genetic background of the mice obtained from the different suppliers, or differences in collagen detection methodologies.38
In addition to examination of lung histology, we selected four proteins as biomarkers for pulmonary injury (TGFβRI, TGFβRII, COX-2 and ACE). Three of these (TGFβRI, RII and COX-2) have previously been identified as markers for lung injury in the bleomycin model system. Bleomycin, a chemotherapeutic agent, induces DNA damage as well as oxidative stress, similar mechanisms as radiation. The time course of injury, however, is compressed to 30–60 days, compared with 120–180 days for radiation injuries. Our study showed radiation-induced reductions in TGFβRI, RII and COX-2 but not in ACE. Studies by others suggest that ACE is biphasically regulated in radiation-damaged lung tissue, where initial loss is attributed to the loss of pulmonary endothelial cells and delayed increase is due to fibrotic remodeling.39,40
It is possible that ACE expression was modified prior to our first time point at 90 days postirradiation. COX-2 levels have been demonstrated to be decreased in lung cells from patients with idiopathic pulmonary fibrosis.41
Prostaglandin E2, a downstream product of COX-2, inhibits fibroblast proliferation and transdifferentiation to the myofibroblast phenotype, suggesting that reduction of COX-2 may be associated with progression of fibrosis.42
TGFβRs have also been reported to have altered expression following lung injury induced by bleomycin.31,32
Khalil et al.
used in situ
immunohistochemistry to identify a transient decrease of TGFβRI expression on alveolar epithelial cells in the early stages following bleomycin-induced injury in rats (day 4–7), believed to correlate with proliferation of these cells in the early repair process.31
This group found no alterations in TGFβRII. However, Zhao et al
., examining overall expression patterns of the receptors, reported increased TGFβRI expression rat lungs following bleomycin treatment for the full time course of their experiments (day 3–12).32
Zhao and coworkers also reported a transient decrease TGFβRII expression during the early reparative stage. We observed a significant transient reduction in TGFβRII expression at 90 days, similar to that previously reported by Zhao.32
We also found a transient reduction in TGFβRI (Alk5 isoform), but this is the opposite of findings in the bleomycin model, where this receptor was reported to have transient increased expression.32
At 90 days postirradiation we found that genistein prevented radiation-induced reduction in the level of TGFβRI expression, but not that of TGFβRII. However, by 180 days postirradiation, all protein expression levels had recovered to control values. Future studies will examine the regulation of TGFβRs in relation to TGF ligands β1, β2 and β3, which are also expressed in lung injury and fibrosis.
In our studies, mice administered the PEG-400 excipient also exhibited an enhancement in 30-day survival (53%) in comparison to the non-treated irradiated mice (23%) animals. The radioprotective properties observed for PEG-400 have previously been demonstrated22,43
and are similar to the protective effects of other excipients such as Emulphor,44
and the polyethoxylated castor oil vehicle Cremophor.46
The modest radioprotective activity of these excipients is believed to be mediated through modulation of the immune system.
Our laboratory has shown that in vivo
genistein prevents acute injuries from radiation.22,47
However, a number of studies have demonstrated in vivo
and in vitro
that genistein may act as a radiosensitizing agent. Genistein increases radiation-induced apoptosis in several cancer cell lines, including human prostate carcinoma cells, leukemic cells, and cervical cancer cells.48–50
In orthotopic prostate tumor transplants in mice, genistein combined with radiation treatment had greater inhibitory effects on prostate tumor growth than radiation alone.51
The differential effect of genistein on cancer cells compared with normal cells may be related to the arrest of cancer cells in the G2
/M phases of the cell cycle.52–54
/M arrest of cancer cells by genistein has been the topic of a number of studies that have identified both the inactivation of nuclear factor-kappa B and the stable activation of ERK1/2 mitogen activated protein kinases as potential mechanisms.52–54
Unlike the G1
and S phases of the cell cycle which have active DNA repair mechanisms, the G2
/M phase of the cell is not associated with DNA repair activity.55
The activity of genistein in sensitizing cells to DNA damaging agents has been shown in cell culture studies to be selective toward cancer cells and not toward normal cells.56
The mechanism(s) by which genistein selectively causes radiosensitization versus radioprotection has not been elicidated.
The relative sensitivity of the lung to radiation-induced damage is hypothesized to be due to the lung’s high oxygen content compared with other organs. While primary radiation injury results in direct damage to cells, secondary effects can arise from the ionization of oxygen, which can form radicals that initiate and propagate chain reactions.57,58
Pneumonitis and pulmonary fibrosis in both humans and animal models are dependent upon the total radiation dose, fraction size, and volume of lung exposure.8,59,60
Although anti-inflammatory treatments (steroidal and non-steroidal) are effective in treating inflammation associated with early radiation-induced pneumonitis, they do not prevent the subsequent fibrosis.61,62
Using TBI in C57BL/6J mice, our results indicate that genistein protects against acute radiation-induced mortality, radiation-induced DNA damage in lung fibroblasts, and some transient radiation-induced alterations in protein expression in the lung. Genistein has a variety of biological properties that may reduce lung injury. These include its anti-inflammatory properties,17,18
and effects on the cell cycle.52,63
Further experiments are required to establish the mechanism(s) responsible for the radioprotective effects of genistein. Additional studies using high dose thoracic radiation will determine whether administration of genistein can prevent radiation-induced pneumonitis and/or fibrotic remodeling in the lung.