The Role Of Oxidative Stress In Radiationinduced Late Effects


A number of factors contribute to the inherent vulnerability of the brain and neural tissue to oxidative stress. The brain represents one of the most metabolically active organs in the body, consuming an inordinate fraction (20%) of the total O2 consumption for its relatively small weight (2%) (134). This leads to a relatively high intracellular production of O2- and other ROS; 2-5% of the O2 consumed in mitochondrial electron transport is converted to O2-. In addition, studies using isolated brain mitochondria indicate that H2O2 production represents about 2% of the total O2 consumed when NADH supplies the reducing equivalents (135). The brain is rich in the more readily oxidizable polyunsat-urated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid, while myelin membrane is a preferential target of ROS due to its composition and high lipid to protein ratio (136). The human brain has high iron content in some brain regions and cells, particularly in oligodendrocytes (137) and in general has high levels of ascorbate. Thus, if tissue injury occurs, the Fe-ascorbate mixture can be a potent prooxidant for brain membranes (138). The brain has a limited ability to perform aerobic glycolysis so it is unusually vulnerable to hypoxia (139). Finally, the brain contains relatively low levels of SOD, catalase and GPx (140) and antioxidants in oligodendrocytes, neurons and endothelial cells (141,142). Oxidative and/or nitrosative stress have been implicated in many neurodegenerative diseases (114,141,143,144). Moreover, increasing and decreasing SOD levels leads to protection (145,146) and sensitization (147) of the CNS to oxidative stress, respectively.

Indirect evidence in support of a role for oxidative stress in radiation-induced CNS injury was presented by Hornsey et al. (148), who postulated that radiation-induced ischemia, seen not only in the CNS but also in other late-responding organs (149), was associated with the development of reperfusion injury (150). Both ROS and RNOS, generated by mitochondria within post-ischemic vascular endothelium (109) are involved in reperfusion injury. Although direct evidence of reperfusion injury was not presented, rats fed with a low-iron diet from 85 days and the Fe-chelating agent, desferrioxamine, from day 120 after local spinal cord irradiation, did exhibit a delay in the onset of ataxia due to white matter necrosis and reduced incidence of lesions after single doses of 25 and 27 Gy X-rays.

More recent data suggest a primary role for chronic oxidative stress and ROS/RNOS in radiation-induced brain injury. As discussed above, initial indirect evidence showed that irradiation of the rat brain inhibited hippocampal neurogenesis, associated with a marked increase in the number and activation status of microglia in the neurogenic zone (45). Subsequent studies showed that inhibiting microglial activation using indomethacin restored hippocampal neurogenesis (63). Indeed, a plot of neurogenesis against activated microglial load for each irradiated rat revealed a negative correlation (r = -0.93) for activated microglial loads of > 1000 cells/dentate gyrus, as compared with a value of approximately 500 in the controls, inferring that the extent of inflammation has a direct role on neurogenesis in the adult rat dentate gyrus.

Direct experimental evidence for radiation-induced oxidative/nitrosative stress has been obtained from studies using neonatal and adult rats and mice. Fukuda et al. (151) treated one hemisphere of postnatal day 8 rats or postnatal day 10 mice with a single dose of 4-12 Gy of 4 MV X-rays. Time-dependent increases in nitrosative stress, assessed in terms of nitrotyrosine formation, were observed in the subventricular zone and the granular cell layer of the dentate gyrus 2-12 h postirradiation. An oxidative stress, evidenced as a significant increase in lipid peroxidation measured using malondialdehyde was noted in the adult male mouse hippocampus 2 weeks after brain irradiation with a single dose of 10 Gy (152). In accompanying in vitro studies using isolated multipotent neural precursor cells derived from the rat hippocampus, Limoli et al. (152) showed that the levels of ROS were significantly elevated when the cells were cultured at low cell density and was associated with elevated proliferation and increased metabolic, primarily mitochondrial activity. The ROS appeared to result from altered mitochondrial function that ultimately compromised the growth rate of the neural precursor cells. At high cell densities, intracellular ROS and oxidative damage were reduced; this was associated with a concomitant increase in MnSOD expression. Irradiation-induced depletion of neural precursor cells assessed in the subgranular zone also led to increased ROS and altered proliferation, confirming the in vitro studies. To further test the role of ROS, mice were treated with the antioxidant a-lipoic acid (LA). LA administration in vivo reduced cell proliferation in both unirradiated and irradiated mice. Indeed, the effect of LA was less marked due to the pronounced reduction of precursor cell numbers observed after irradiation. Of note, LA treatment in irradiated mice lowered malondialdehyde levels in hippocampal tissue, supporting the active role of radiation-induced oxidative stress in radiation-induced brain injury.

In more recent studies, acute (0-24 h) and chronic (3-33 days) changes in apoptosis and ROS were measured in irradiated neural precursor cells (153). Irradiating neural precursor cells led to an acute dose-dependent increase in apoptosis accompanied by an increase in ROS. Of note, this oxidative stress persisted over the chronic period of assessment. In vivo studies using wild type and Trp53-null mice indicated a reduction in radiation-induced apoptosis in the latter group, suggesting that the apoptotic and ROS responses might be linked to Trp53-mediated regulation of cell cycle control and stress-activated pathways. The presence of oxidative stress was again observed in terms of a marked increase in the levels of malondialdehyde determined 1 week after a single dose of 10 Gy to the mouse brain (153). More recently, Rola et al. (154) have reported a chronic inflammatory response in the mouse dentate subgranular zone 9 months following high-LET brain irradiation; expression of the CCR2 receptor, important in neuroinflammation (155,156), increased in the irradiated brains as compared to the sham-irradiated control brains.

Experimental data describing oxidative stress in the irradiated spinal cord are more limited. Increased expression of Hmox-1, a common marker of oxidative stress, has been observed in the irradiated rat spinal cord prior to the onset of myelopathy (16). Moreover, a time- and dose-dependent increase in hypoxia has been observed in the irradiated rat spinal cord prior to the onset of white matter necrosis (157). Hypoxia has been shown to lead to increased ROS/RNOS production in various cell types (158-160), due to increased ROS/RNOS generation and reduced antioxidant and/or antioxidant enzyme production (160). Similarly, increased expression of the redox-regulated gene product PAI-1 (161) has been reported prior to radiation-induced necrosis (162).


Oxidative stress clearly plays a role in chronic renal failure (163-165). Radiation nephropathy is characterized by chronic progressive renal dysfunction, glomeruloscle-rosis and tubulointerstitial fibrosis (77). The accepted threshold dose of photon irradiation that will cause radiation nephropathy is exposure of both kidneys to 23 Gy total dose, fractionated in 20 doses over 4 weeks (166). If only one kidney is irradiated with a threshold or higher dose, radiation injury will occur in that kidney, but kidney failure from radiation nephropathy per se will not occur. However, the unirradiated kidney is likely to become damaged from the renin-mediated hypertension that occurs because of the severe unilateral renal scarring (167).

In the case of radiation nephropathy after bone marrow transplantation (BMT), a 10-Gy single dose of X-rays to the kidneys will cause this form of radiation nephropathy, as will 14 Gy, fractionated over 3 days (168). As for the radioisotope-induced radiation nephropa-thy, the exact delivered doses are not always well defined. In the case of the rhenium conjugate used for radioimmunotherapy, the total kidney dose from the radionuclide is estimated at 7 Gy (169). This dose would not in itself be sufficient for kidney injury, but, because it was added to 12-Gy TBI in the patients of that report, it provides an additional nephrotoxic effect.

Chronic oxidative stress has been observed in the irradiated kidney. Robbins et al. (170) adopted an indirect approach using immunohistochemical detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage (171). Sham-irradiated kidneys showed little evidence of DNA oxidation over the 24-week experimental period. In marked contrast, localized kidney irradiation led to a marked, dose-independent increase in glomerular and tubular cell DNA oxidation, evident at the first time point studied, i.e. 4 weeks after irradiation, that was maintained for up to 24 weeks postirradiation. Since the repair enzymes for 8-OHdG are present in the rat kidney (172), the failure of the kidney cell 8-OHdG staining to decrease suggested the presence of a chronic, persistent oxidative stress in the irradiated kidney for up to 24 weeks postirradiation.


Increased oxidative stress is a significant part of the pathogenesis of chronic lung disease, including obstructive lung disease and idiopathic pulmonary fibrosis (173,174). The lungs are particularly sensitive to irradiation. Radiation-induced lung injury is related to both dose and volume effects. The TD5/5 for one-third, two-thirds and whole lung is 45, 30 and 17.5 Gy, respectively. Radiation-induced lung injury is characterized by an acute pneumonitic phase that is followed by a phase of chronic inflammation and fibrosis that develops months or years after irradiation (175). Radiation-induced pneu-monitis after unilateral irradiation of the rat lung leads to increased expression of NOS

and 'NO production (176). More recent studies not only identified iNOS as the major source of 'NO, but also identified radiation-induced nitrosative stress in the rat and mouse lung, evidenced by the presence of nitrotyrosine in the alveolar epithelium, macrophages and vascular endothelium (177,178). These findings have been confirmed in patients. Oxidative stress assessed using systemic markers of lipid peroxidation or by increased oxidized methionine in bronchoalveolar lavage fluid has been observed during and after the completion of radiation therapy in lung cancer patients (179-181).

Additional data in support of a pathogenic role for chronic oxidative stress in radiation-induced lung injury has come from studies in the rat (182). Hypoxia was identified in the rat lung 6 weeks after a single dose of 28 Gy using the hypoxia marker pimonidazole, and much earlier than the onset of functional or histopathologic changes. This hypoxia became progressively more severe, such that at 6 months postirradiation it was associated with a significant increase in macrophage activity, fibrosis and increased breathing rate. Immuno-histochemical evaluation revealed increases in TGF-P, VEGF and CD-31 endothelial cell markers, suggesting a hypoxia-mediated activation of profibrotic and proangiogenic pathways. Additional evidence in support of a radiation-induced chronic oxidative stress in the lung comes from recent studies in which increased lipid peroxidation, assessed in terms of malondialdehyde levels, was determined in the lungs of mice 15-20 weeks postirradiation (183). These increases in oxidative stress were not observed in irradiated lungs of transgenic mice overexpressing EC-SOD, which were protected against radiation-induced lung injury. Protection against radiation-induced acute and late lung injury has also been observed in mice overexpressing a transgene for human MnSOD (184,185).


Accidental radiation overexposure to the skin and underlying subcutaneous tissues can lead to severe lesions resulting in extensive fibronecrotic tissues. In high-dose radiation accidents, fibrosis is usually the result of scarring following tissue necrosis (79). Moreover, in clinical practice radiation-induced fibrosis of the skin and underlying soft tissues can occur months to years after therapeutic irradiation (186). While there are currently no data directly demonstrating the presence of chronic oxidative stress in irradiated skin and subcutaneous tissues, indirect evidence in support of this hypothesis has come from studies using antioxidant-based interventions. Thus, administration of liposomal CuZnSOD and MnSOD 6 months after irradiation in an experimental model of radiation-induced fibrosis reversed the radiation-induced fibrosis and resulted in the regeneration of normal tissue in a zone of well-established postirradiation fibrosis (187). Similar findings were observed clinically (188). A striking regression of radiation-induced fibrosis has been observed experimentally and clinically using a combination of pentoxifylline and the antioxidant a-tocopherol (189,190).

More recently, Delanian et al. (191) reported on a retrospective series of 44 breast cancer patients with superficial progressive radiation-induced fibrotic lesions. Patients received a combination of pentoxifylline and a-tocopherol twice daily for either 6-12 months or 24-48 months. Patients were assessed in terms of reduction in the size of the fibrotic region and in the global score of late injury. Of note, both treatment groups showed significant improvements in the extent of fibrosis and in the severity of late injury. The regression in radiation-induced fibrosis appeared exponential, with a 67% maximum response after a mean of 2 years. Importantly, there was a risk of a rebound effect if the treatment was too short, i.e. less than 3 years. This antioxidant-based modulation of radiation-induced fibrosis appears to reflect alterations in the phenotype of irradiated fibroblasts (192). Reduced TGF-^1 and anti-collagenase TIMP expression was observed, with an increase in endogenous MnSOD. In myofibroblasts cultured from pig skin that had developed radiation-induced fibrosis, a reversion of the myofibroblast phenotype to a normal fibroblast phenotype was observed following treatment with CuZnSOD (193).

These data indicate that radiation may activate local mediators and thereby initiate continuous generation of ROS, causing chronic oxidative stress. In addition, there is a wealth of indirect data from studies using antioxidant/anti-inflammatory-based approaches that show prevention or mitigation of radiation-induced late normal tissue injury (15,194-196). Indeed, the most successful interventional approach to reduce the severity of late radiation-induced injury, namely blockade of the renin-angiotensin system (RAS), reflects, in part, inhibition of chronic oxidative stress (197).

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