The objective of this study was to characterize the acute molecular response to whole-body proton irradiation in the absence and presence of dietary antioxidants in mice in vivo. These data confirm the high-energy proton-induced gene expression of classical markers of apoptosis, including the activation of the downstream effectors caspase-3 and PARP-1 (), in the bone marrow of mice. In the presence of the dietary antioxidants, total bone marrow mRNA and protein expression of apoptosis-related genes were decreased compared to the expression profiles in the irradiated mice not receiving the antioxidant formulation. It is speculated that the antioxidants are altering mRNA and protein levels by reducing the generation of reactive oxygen species and total cellular antioxidant status (or cellular oxidative stress). A general scheme of the apoptosis-related genes and their potential roles in the mitochondria after ionizing radiation exposure and in the presence of antioxidants is illustrated in .
Potential role of antioxidants in the mitochondria in response to proton radiation exposure.
Significant increases in BAX, Bcl2 and Bcl-xL expression in proton-irradiated mice suggest mitochondrial dysfunction and activation of the cell-intrinsic apoptotic pathway as opposed to the cell-extrinsic pathway involving death receptors (14
). BAX, Bcl2 and Bcl-xL, all members of the Bcl superfamily, are downstream target genes of the tumor suppressor gene tp53. BAX is a pro-apoptosis gene. In response to cellular stress BAX relocates to the surface of the mitochondria where the anti-apoptosis proteins, including Bcl2 and Bcl-xL, are located. The interaction between the anti- and pro-apoptosis proteins forms pores in the mitochondria, causing the release of cytochrome c, which in turn activates the caspase cascade (). A decrease in mRNA levels of BAX is observed in mice fed the antioxidant diet and exposed to proton radiation (). The proton radiation-induced increases in BAX mRNA levels are consistent with previous findings of γ-radiation-induced increases of BAX gene expression in hematopoietic tissues (15
). Our findings show increased expression of both Bcl2 and Bcl-xL proteins, which prevent the release of cytochrome c (17
), in the presence of dietary antioxidants ( and ).
Caspase-9 mRNA expression is significantly upregulated after proton irradiation and downregulated in the presence of antioxidants after proton irradiation (). Caspase-9 is activated upon cytochrome c release from the mitochondria, and the activated complex cleaves or activates effector caspase, caspase-3. Caspase-3 cleavage is significantly induced after proton irradiation (), and protein detection of the activated form of caspase-3 is decreased in the presence of dietary antioxidants ().
Caspase 8 is known as an initiator caspase of the extrinsic pathway involving transmembrane death receptors. Upon ligation, the death receptors activate caspase-8, initiating effector caspase signaling (19
). caspase-8 mRNA expression was increased after radiation exposure (); however, the active form of caspase-8 was not measured in this study. Expression levels of pro-caspase 8 were significantly decreased after radiation exposure in the presence of dietary antioxidants, suggesting an abrogating effect on this signaling pathway. NFκB suppresses death receptor signaling (19
), and elevated NFκB mRNA levels were observed after radiation exposure (), contributing to the extrinsic pathway.
By definition, antioxidants scavenge free radicals. The antioxidants used in this study vary widely in their chemical structure, and this may contribute to other biochemical functions and cellular processes that anti-oxidant treatments have been shown to hinder, including gene expression of apoptosis-related genes (20
), as reported here. It is generally accepted that ionizing radiation results in oxidative stress (22
) within the biological system and the generation of reactive oxygen species (ROS) (23
). ROS are generated in the mitochondria by the respiratory chain, at the level of coenzyme Q10 (one component of the antioxidant mix used in this study). Oxidative stress and ROS are considered to induce apoptotic cell death through direct interaction with DNA or the initiation of the intrinsic pathway for cell death. The latter begins in the mitochondria where ROS cause mitochondrial outer membrane permeabilization (23
) and subsequent release of cytochrome c and other proteins. Regulators of the permeabilization include members of the Bcl2 family Bax and Bak, while anti-apoptotic Bcl2 and Bcl-xL inhibit protein release. Cytochrome c release from the mitochondria into the cytoplasm initiates the caspase cascade of events, as mentioned above.
Antioxidant supplementation has been shown to prevent radiation-induced oxidative stress (24
). We speculate that antioxidant supplementation protects against radiation-induced oxidative stress by either blocking ROS production or neutralizing mitochondrial ROS in animals exposed to 1 Gy proton radiation. The prevention or scavenging of radiation-induced ROS may reduce mitochondrial membrane permeabilization and the levels of expression of pro-apoptosis-related genes that are intimately involved in the mitochondria-associated intrinsic death pathway. These results using small molecule antioxidants are consistent with those of Bai et al.
), who reported that antioxidant enzymes target mitochondrial ROS, affecting apoptotic stimuli.
It is expected that the prevention of radiation-induced apoptosis will result in enhanced survival of the irradiated cells. Because these cells have evaded apoptosis or cell death, the genomic stability of these cells might be considered questionable. The long-term consequences associated with their survival, however, are unknown. It has been hypothesized that radioprotectors may lead to the rescue of damaged cells that could play a role in tumor initiation or progression and ultimately result in increased carcinogenesis (27
). While this is a reasonable hypothesis, the evidence thus far does not suggest that the use of radioprotective agents increases radiation-induced carcinogenesis. Most research on radioprotectors and carcinogenesis has been performed with WR2721/WR1065 (also known as amifostine). Amifostine has been shown to have radioprotective (28
), antimutagenic (28
) and anticarcinogenic (29
) properties in numerous in vivo
and in vitro
systems, as has been reviewed (30
) by Grdina et al. Because the abilities of the active compound are quite different for cytoprotection and anticarcinogenic activities, this has suggested that the mechanisms of action and pathways that are involved in rescuing cells from radiation-induced cytotoxic effects are quite different from those involved in protection against radiation carcinogenesis (30
). Other radioprotectors that inhibit both radiation-induced cell killing and carcinogenesis include the soybean-derived Bowman-Birk inhibitor and the antioxidant combination used in the studies reported here (2
). These results suggest that for these agents, effects on the genes/pathways leading to the rescue of cells destined to die from the cell killing effects of radiation do not have enhancing effects, and may have suppressing effects, on the genes/pathways involved in radiation-induced carcinogenesis. It can be speculated that abrogation of radiation-induced expression of proapoptosis genes could increase or decrease carcinogenic potential, but the long-term effects of radiation induced modification of gene expression are unknown and warrant further research.
The data presented here suggest that proton radiation induces varying molecular mechanisms that simultaneously promote and suppress programmed cell death. Antioxidant supplementation during proton irradiation significantly decreases expression levels of pro-apoptosis genes conferring protection against apoptosis in cells of hematopoietic origin.