INDUCTION OF CELL DEATH THROUGH ALTERATION OF OXIDANTS AND ANTIOXIDANTS IN LUNG EPITHELIAL CELLS EXPOSED TO HIGH ENERGY PROTONS

Radiation affects several cellular and molecular processes, including double strand breakage and modifications of sugar moieties and bases. In outer space, protons are the primary radiation source that poses a range of potential health risks to astronauts. On the other hand, the use of proton irradiation for tumor radiation therapy is increasing, as it largely spares healthy tissues while killing tumor tissues. Although radiation-related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton irradiation remain poorly understood. Therefore, in this study, we irradiated rat lung epithelial cells with different doses of protons and investigated their effects on cell proliferation and death. Our data show an inhibition of cell proliferation in proton-irradiated cells with a significant dose-dependent activation and repression of reactive oxygen species and antioxidants glutathione and superoxide dismutase, respectively, compared with control cells. In addition, the activities of apoptosis-related genes such as caspase-3 and -8 were induced in a dose-dependent manner with corresponding increased levels of DNA fragmentation in proton-irradiated cells compared with control cells. Together, our results show that proton irradiation alters oxidant and antioxidant levels in cells to activate the apoptotic pathway for cell death.


INTRODUCTION
The mechanism by which radiation causes damage to human tissue is by ionization of atoms in the material. Radiation is known to interfere with cellular functions at all levels of cell organization. Studies of people exposed to high doses of radiation have shown that there is a risk of cancer induction associated with high doses. The specific types of cancers associated with radiation exposure include leukemia, multiple myeloma, breast cancer, lung cancer, and skin cancer. Animals exposed to 55-MeV protons had a high incidence of malignant brain tumors (1). Accumulating evidence suggests that radiation-induced genomic instability is a non targeted phenomenon triggered by radiation that may initiate and likely contribute to radiation induced carcinogenesis (2). In outer space, protons are the primary radiation source. They pose a range of potential health risks to astronauts including work performance, psychological, as well as somatic functions (3). Literature on this subject has shown that space radiation induces oxidative stress mediated cell damage in astronauts after space flight (4). On the other hand, proton therapy is the most precise, efficient, and advanced form of radiation treatment today. It primarily irradiates the tumor site, leaving surrounding healthy tissue and organs intact. Cell death induced by proton beam has also been identified as apoptosis (5). Irradiation studies on neural cells showed the depletion of precursor cells in vivo, and reductions of these critical cells are believed to impair neurogenesis and cognition (6).
Radiation induced DNA damage investigation is one of the most important areas in modern biology, but still the information available on the effects of ionizing radiations, particularly protons, is very limited. Our recently published and earlier observation on proton irradiated mouse brain showed an alteration of oxidative stress mediated apoptosis inducing genes and differential expression pattern of DNA damage and oxidative stress related genes (7,8). In this report, we have developed an in vitro system using cultured rat lung epithelial cells (LE) and have studied proton mediated cell killing. Our observation showed an increased level of reactive oxygen species (ROS) and lipid peroxidation (LPO) followed by inhibition of antioxidants, glutathione (GSH) and superoxide dismutase (SOD) in proton irradiated cells as compared to control cells. In addition, a significant activation of cell death related genes such as caspase 3 and 8 was detected in these cells. Together, these observations suggest that both in-vivo and invitro model systems, proton radiation causes similar effects by inducing oxidative stress which in turn activates signaling cascade for DNA and cellular damage.

Cell line and Proton Exposure
Rat lung epithelial cells (RL 65, CRL-10354) were purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM with 10% FBS, 100 IU/ml of penicillin, and 100 μg/ml of streptomycin and incubated at 37 0 C in a humidified chamber with 5% CO 2 . Exponentially growing LE cells were split and reseeded 1 day prior to irradiation. LE cells were irradiated with 250 MeV protons at different doses (0.1Gy, 1Gy, 2Gy and 4Gy) at Loma Linda Radiation facility (California, USA), cultured and harvested at different time points depending on experimental procedure. For comparative purposes, control cells were cultured similarly and harvested along with irradiated cells.

Cell Viability Assay
The MTT system is a simple, accurate, reproducible means of measuring the activity of living cells via mitochondrial dehydrogenase activity. The key component is 3-[4, 5dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide or MTT. Solutions of MTT solubilized in tissue culture media or balanced salt solutions, without phenol red, are yellowish in color. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple MTT formazan crystals which are insoluble in aqueous solutions. The crystals can be dissolved in acidified isopropanol. The resulting purple solution is spectrophotometrically measured. An increase in cell number results in an increase in the amount of MTT formazan formed and an increase in absorbance. As described earlier, the cytotoxicity assay was performed using MTT (9). LE cells grown overnight were irradiated at different doses of protons and cultured for 36h. The irradiated LE cells were washed with PBS and MTT was added to a final concentration of 125µg/ml and incubation continued for another 3h. The formazan formed inside the cells were extracted using acidic methanol and the absorbance was measured at 570 nm. Live and dead cell assay was performed essentially as described elsewhere (9). Briefly, 10 5 LE cells were cultured for 24h and irradiated with different doses of protons and incubated for another 24h. The irradiated cells were stained with 5µM ethidium homodimer and 5µM calcein-AM (Molecular Probes, Eugene, OR) and incubated for 1h at 37 0 C. The stained cells were analyzed under fluorescent microscope (Zeiss, Germany) and photographed.

Detection of Reactive Oxygen Species (ROS)
The measurement of intracellular reactive oxygen species (ROS) was performed as described earlier (10). Briefly, equal numbers of rat LE cells (2000 cells/well) were seeded in 96-well plates and grown for 24h. The cells were then incubated with 10 μM of H 2 DCF-DA for 3h, washed with PBS, exposed to different doses of protons and the intensity of fluorescence was measured at excitation and emission of wavelength at 485/527 nm, respectively. The readings were taken immediately after irradiation and continued up to 3h. The 2.5h of post irradiated readings were shown in fig 2A as expressed fluorescence units.

Assay for Lipid Peroxidation (LPO)
Proton induced lipid peroxidation was determined using a kit from Cayman Chemicals as described earlier (11). Equal numbers of LE cells (4×10 5 cells/well) were seeded in 6-well plates and grown for 24h. Following incubation, cells were washed with PBS and exposed to different doses of protons and incubated for 12h. The cells were then scraped with PBS and sonicated. Fifty microgram of cell lysate and methanol were mixed and centrifuged after adding pre cooled chloroform at 1500× g for 10min. The supernatant containing hydro peroxides was collected and used for the estimation of thiobarituric acid reactive malondialdehyde (MDA). The chromogen formed was detected at a wavelength of 500 nm.

Detection of Glutathione
Glutathione is the key antioxidant present in most of the cells (12). The intracellular reduced GSH activity was measured by glutathione assay kit as per the instructions provided by the manufacturer. In brief, LE cells (4 × 10 5 cells/well) were seeded in a 6-well plate and grown for 24h. Next, cells were irradiated with different doses of protons and incubation continued for 12h. Then, the cells were scraped and homogenized using PBS. Fifty microgram of protein was deproteinized using 5% 5sulfosalicylic acid dihydrate solution and sodium carbonate (400 mM) followed by 1:8 dilutions with phosphate-EDTA buffer and incubated for 10 minutes at room temperature.
The supernatant was then treated with 5, 5-di-thiobis (2-nitrobenzoic acid; DTNB) and incubation continued for another 10 min. The GSH activity was measured at 415 nm absorbance.

Superoxide Dismutase Assay
The assay was performed by using Superoxide Dismutase (SOD) Kit from Trevigen, Inc Gaithersburg, MD, USA (Cat # 7500-100-K). Fifty microgram of protein extracts were used to assay total SOD activities using manufacturer's protocol. Briefly, SOD reaction buffer was mixed with xanthine solution followed by NBT solution and then the sample proteins isolated from 12h after proton irradiation were added and set the absorbance to zero at 550 nm. Finally, XOD solution was added to each sample and readings were taken at 550 nm every 30 seconds for a period of 5 minutes. The total SOD activity was calculated based on the manufacturer's formula.

Western Blotting
Whole cell extracts were prepared from different time points of proton irradiated and control cells using mammalian cell extraction buffer (Biovision; Mountain View, CA) as described previously (7). Equal amounts of proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene diflouride membrane. The membrane was blocked in 5% non fat dry milk powder in PBS containing 0.1% IGEPAL, probed with appropriate primary antibody followed by secondary antibody conjugated with horse radish peroxidase and developed using Pierce detection solution (Thermo Scientific, Rockford, IL, USA). The following antibodies were purchased from santacruz (Santa Cruz, CA) and used; caspase 3 (SC-7272), caspase 8 (SC-7890), SOD-1 (SC-11407), SOD-2 (SC-18503) and β actin (Sigma; cat # A5441).

Caspase-3 and Caspase-8 Activity Assay
Cleavage activities of caspase-3 and 8 substrates DEVD-AFC and IETD-AFC were measured according to manufacturer's protocol (Santa Cruz Biotechnology Inc). Briefly, protein extracts were made from 24h of post proton irradiated and control cells followed by estimation using coomassie plus protein assay reagent (Thermo fisher; cat # 1856210). DEVD-AFC and IETD-AFC substrates were added to fifty microgram of protein extract for detecting caspase-3 and caspase-8, respectively and incubated for 1h at 37 0 C. The formation of free AFC in the extract was measured at an excitation wavelength of 400 nm and an emission wavelength of 495 nm. The values of experimental samples were compared with control and expressed as fluorescence units.

DNA Isolation and DNA Fragmentation
Genomic DNA was isolated from proton radiated (1, 2 and 4Gy) and control cells using Quick Apoptotic DNA ladder kit (Invitrogen; Cat # SKU-KHO1021) according to the procedure specified by manufacturer. Briefly, equal number of cells from 24h of post irradiated and control were homogenized in TE buffer followed by mixing with Enzyme A solution and incubated at 37 0 C for 10 minutes. Enzyme B solution was added to the enzyme A mix and incubated for additional 30 minutes at 50 0 C. To this, one tenth volume of ammonium acetate and 2.5fold cold ethanol were added and precipitated at -20 0 C for 15 minutes followed by centrifugation to get the DNA pellet. The pellet was washed with 70% cold ethanol and centrifuged again. Finally, the DNA was airdried and resuspended in DNA suspension buffer and analyzed on 1.2 % agarose gel.

Statistical Analysis
Data were expressed as mean ± SD and statistical significance was analyzed by student's t-test. A p-value of < 0.05 was considered statistically significant.

Proton Irradiation Inhibits Cell Viability in
Lung Epithelial Cells. Previous observations from our group have shown the induction of DNA and tissue damage in 2Gy proton irradiated mouse brain tissues (7,8). Therefore, in this study, we tested the effect of protons on the in vitro system of rat LE cells. Here, we show a significant dose dependent inhibition of cell proliferation in proton irradiated cells as compared to control cells which is evident by standard MTT dye uptake cell viability assay (Fig 1A). Notably, LE cells exposed to a lower dose 0.1Gy do not show any change in cell proliferation as compared to control untreated cells. To reconfirm cell viability, a live-dead cell assay was performed which showed the similar effect with an increased numbers of dead cells in proton irradiated cells as compared to control cells in a dose dependent manner ( Figure 1B).

Activation of ROS and LPO in Proton
Irradiated Cells. Since we observed an inhibition of cell proliferation in proton irradiated cells, we were interested in checking whether proton irradiation alters oxidative stress to inhibit cell proliferation. As shown in Fig 2A, proton irradiation activated reactive oxygen species significantly in a dose dependent manner as compared to control cells. For example, ROS levels were higher with 3 folds at 4Gy, 2.5 folds at 2Gy and 1.8 folds at 1Gy than control cells. However, a lower dose of 0.1Gy does not affect ROS production. It has been shown in the literature that lipid peroxidation, a standard biomarker for oxidative stress, is activated during external stress (12). Therefore, we measured lipid peroxidation levels at different time points of proton irradiated cells and found a dose dependent increased level of LPO in these cells which directly correlates with increased ROS levels observed in proton irradiated condition (Fig 2B).

Inhibition of Antioxidants in Proton
Irradiated Cells. The presence of many antioxidants has been reported in cells as protective mechanism during oxidative stress and apoptotic cell death (13). Glutathione and Superoxide dismutase are the major antioxidants in the cells to balance ROS levels to maintain normal cellular functions when cells are under external stress (14). Since alteration of oxidant level has been observed in proton irradiated cells, antioxidant levels were also analyzed. A significant dose dependent inhibition of GSH and SOD activities was detected in proton irradiated cells as compared to control cells. (Fig  3 A & B). Notably, a 50% and 80% of reduction of GSH and SOD activities were detected respectively at 4Gy proton irradiated cells than control. In addition, fig 3C shows the dose dependent reduction of SOD-1 and 2 proteins level at 12h of post irradiated cells as compared to control cells.

Proton Irradiation Induce Caspase 3 and 8 in LE Cells
. DNA damage is mainly caused by external factors, particularly radiation and chemicals (15). Our group has used external stress inducing agents in in vitro system and shown an induction of oxidative stress mediated cell death through activation of caspase-3 and 8 (16,12). Here, we performed the same strategy in proton irradiated cells to verify whether caspase levels are affected by proton irradiation. Fig 4A shows a significantly increased level of caspase 3 activities at higher proton doses, 2 and 4Gy as compared to control and lower dose 0.1Gy. Furthermore, the activation of caspase-3 is dependent on the early activation of either caspase-8 or caspase-9. So, we continued our investigation on caspase-8 activity and showed a dose dependent increase of caspase-8 activity at 2Gy (2fold) and 4Gy (3fold) proton irradiated cells as compared to control cells (Fig 4B). Our protein data for caspase 3 and -8 also show an increased level of these proteins in 2 and 4Gy irradiated cells as compared to control cells ( Fig  4C).

Proton Induced DNA Fragmentation in LE
Cells. It has long been known that radiation induces DNA damage. Also, our recently published report on irradiated mouse brain showed significant DNA damage as compared to the control brain (7). DNA fragmentation is a key feature of programmed cell death and the process is characterized by the activation of endogenous endonucleases with subsequent cleavage of chromatin DNA into internucleosomal fragments of 180 base pairs and multiples thereof (15). There are many methods to assess the DNA fragmentation caused by apoptosis event and the most standard technique involves detection of DNA ladders using agarose gel electrophoresis.

DISCUSSION
Proton radiation (PR) therapy offers a number of potential advantages over conventional (photon) gamma-radiation (GR) therapy for cancer, due to a more localized delivery of the radiation dose. It is generally assumed that the relative biological effectiveness (RBE) of protons is 1.1 cobalt-gray-equivalents (CGE; sometimes referred to as Gray equivalents or GyE). At hospital-based proton facilities, a dose of 1.8 to 2 CGE is often used per fraction, with one fraction delivered per day over a period of 5 days/week for 5-7 weeks, depending up on the type and location of the tumor, as well as other considerations. Thus, the dose of 2Gy used in the present study is approximately equivalent to one fraction of proton radiation delivered during therapy. However, it should be noted that although the total doses delivered to the intended target volume during treatment are much higher than used here, normal cells distant from the target may well be exposed to 0.1-4Gy during or by the end of therapy. For example, at Loma Linda University Medical Center, there are seven proton energies used for patient treatment and all are within the range of 100-250 MeV. Protons are also the most abundant type of particles encountered by astronauts during missions in both the Low Earth Orbit (LEO) and to the Moon or Mars (16). The dose received by astronauts during a LEO mission that lasts for several months can be as high as 0.1 Sv. In space, proton energies have a much wider range and dependency on certain conditions. For example, proton energies could be up to several 100 MeV during a solar particle event (SPE) and up to several 1,000 MeV in galactic cosmic rays (GCR) (17). Research on radiation has been ongoing for several decades but the information available on protons is minimal. We currently lack critical knowledge of molecular mechanism of proton radiation, to assess the human radiation exposure, and this is a high priority as it would enable better determination of health risks.
Recently, published reports on a mouse system showed an activation of DNA damage and apoptosis related genes in 2Gy proton irradiated brain tissues than control brain (7). Therefore, in this report, we extended our radiation studies using different doses of protons in an in-vitro cell line system, particularly rat lung epithelial cells and investigated radiation induced oxidative stress followed by cell death.
Our cell viability observations from this study showed that proton irradiation inhibits cell proliferation in a dose dependent manner in LE cells (Fig 1). Several reports have shown that ROS could be activated through various stressors such as uranium, tobacco smoke, carbon nanotubes, high glucose, TNF-α, chemicals, radiation etc. and the elevated levels of ROS regulates a broad array of signal transduction pathways that regulate various biological processes including gene expression, cell growth, differentiation, and apoptosis (18,19). In agreement with the above mentioned ROS activation, our data from this study also showed a dose dependent activation of ROS in proton irradiated cells than control cells (Fig  2A).
In biological systems, oxidant and antioxidant levels are maintained in a balanced state. For example, when cells are under stress, antioxidants are produced at higher concentration to neutralize the ROS level for normal cellular activities. Literature evidence also suggests that over-expression of SOD protects cells from ROS mediated apoptosis (20,21). Here, the current data from proton irradiated cells showed an inhibition of antioxidant levels as compared to control cells suggesting that proton irradiation blocks the production of antioxidants through an unidentified mechanism in order to kill the irradiated cells.
The ultimate effect of radiation is to induce DNA damage and cell death (14). A recent report showed that cell death induced by proton is apoptosis rather than necrosis or autophagy and the apoptosis process are mediated through the activation of caspases (6). Very recently, we also showed that mice exposed to 2Gy proton induce caspase 3 and 8 activation, fragmented DNA and significant tissue damage with altered expression of DNA damage and oxidative stress signaling genes as compared to control brain tissues (7,8). Consistent with our earlier in vivo observation, current in vitro cell line data also show a similar effect with an induction of caspase 3 and 8 and significant levels of fragmented DNA in proton irradiated cells than control cells (Fig 4A & B), which confirms that proton irradiated cells induce apoptotic pathway through activation of caspase3 and -8 for cell death. Even though we detect 2-3 fold increase in caspase activity at 4Gy irradiated cells, the caspase 3 proteins seems to be increased much more from 12h to 24h of 4Gy irradiated cells. At present, we do not know why there is a dramatic increase in caspase 3 at these time points. In addition, apoptotic ladder study showed more DNA fragmentation at 4Gy than control and other lower doses suggesting that the DNA damage followed by their effects on cell death event is dependent on doses, possibly to the exposure time. It is important to note here that our DNA fragmentation and caspase activation data correlate with cell viability data where we observed more dead cells at 24h of 4Gy proton irradiation. Studies on proton mediated cellular signaling pathways such as NF B, Ap-1 etc and their effect on oxidative stress induced cell death is underway to dissect the exact molecular mechanism of protons mediated cell killing.
Taken together, our findings show that there is an involvement of oxidative stress pathway leading to functional activation of cell death related caspase 3 and 8 following proton exposure which complements the response triggered by DNA damage. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current proton therapy protocols.

Fig. 1. Proton Exposure Inhibits Cell Viability. A)
Equal numbers of rat LE cells were seeded and grown for 24h. Cells were then exposed to different doses of proton (0.1, 1, 2 and 4Gy) and cultured for 36h. The cell viability was assayed based on MTT dye uptake (absorbance at 570nm) and the values were calculated based on control. The experiment was carried out in triplicate and the average values with SD are shown. B) LE cells were seeded equally and exposed to proton (1, 2 and 4Gy), cultured for 24h and live and dead cells were visualized based on dye uptake method and photographed. Percentage of live cells was shown below.   3. Inhibition of Antioxidants in Proton Exposed LE Cells. A) Proteins were extracted from 12h of post proton irradiated and control LE cells and 50μg of total proteins were used to assay glutathione activity as described in methods. B) Superoxide dismutase activity was assayed by mixing SOD reaction buffer, xanthine, NBT solution with 50μg of proteins isolated from 12h of post irradiated (different doses of protons) and control LE cells using manufacturer's formula. Both glutathione and SOD results indicated are the mean standard deviation of three independent experiments. C) Protein extracts were made from 12h of post irradiated (different doses) and control cells and analyzed for SOD-1 and SOD-2 proteins using specific antibodies. The β-actin was used as internal loading control and ns represents nonspecific band.

Fig. 4. Induction of Caspase-3 and Caspase-8 Activities in Proton Exposed Cells. A & B)
Protein extracts isolated from 24h of post irradiated (0.1, 1, 2 and 4Gy) and control cells were mixed with DEVD-AFC and IETD-AFC for caspase-3 and caspase-8 activity, respectively and the formation of free AFC in the mix was measured at an excitation wavelength of 400nm and an emission of 495nm. The experimental values were compared with control and expressed as fluorescence units. Values are mean SD of three independent experiments. C) Protein extracts were made from 12 and 24h of 2Gy and 4Gy irradiated and control cells and analyzed for caspase3 and caspase 8 proteins using specific antibodies. The β-actin was used as internal loading control.