Delta-Tocotrienol Protects Mice from Radiation-Induced Gastrointestinal Injury Author(s): Xiang Hong Li, Sanchita P. Ghosh, Cam T. Ha, Dadin Fu, Thomas B. Elliott, David L. Bolduc, Vilmar Villa, Mark H. Whitnall, Michael R. Landauer and Mang Xiao Source: Radiation Research, 180(6):649-657. 2013. Published By: Radiation Research Society DOI: http://dx.doi.org/10.1667/RR13398.1 URL: http://www.bioone.org/doi/full/10.1667/RR13398.1

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RADIATION RESEARCH

180, 649–657 (2013)

0033-7587/13 $15.00 Ó2013 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR13398.1

Delta-Tocotrienol Protects Mice from Radiation-Induced Gastrointestinal Injury Xiang Hong Li,a,1 Sanchita P. Ghosh,a,1 Cam T. Ha,a Dadin Fu,a Thomas B. Elliott,b David L. Bolduc,a Vilmar Villa,a Mark H. Whitnall,a Michael R. Landauera and Mang Xiaoa,2 a

Radiation Countermeasures Program, b Combined Injury Program, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, Maryland

INTRODUCTION Li, X. H., Ghosh, S. P., Ha, C. T., Fu, D., Elliott, T. B., Bolduc, D. L., Villa, V., Whitnall, M. H., Landauer, M. R. and Xiao, M. Delta-Tocotrienol Protects Mice from RadiationInduced Gastrointestinal Injury. Radiat. Res. 180, 649–657 (2013).

Exposure to ionizing radiation causes damage to DNA, protein and lipids in mammalian cells, with subsequent cell cycle checkpoint arrest, apoptosis, senescence and/or necrosis. The hematopoietic and gastrointestinal (GI) systems are among the most radiation-sensitive organs (1– 3). A radiation dose above 1 Gy in humans poses a risk of destruction of the bone marrow and damage to the hematopoietic system, leading to decreases in blood cell and platelet counts, long-term compromised immune function and increased susceptibility to infection and internal hemorrhage (4, 5). High-dose (10 Gy or more) total-body irradiation (TBI) in experimental mice can result in acute generalized GI syndrome with loss of intestinal crypts, damage to crypt stem cells, and breakdown of the GI mucosal barrier, leading to animal death (3, 6). The small intestine is a constantly renewing tissue, replacing cells that are lost into the lumen of the intestine. This renewal is achieved by the production of new cells in the crypts by the stem cells and their progeny, arranged in an amplifying transit lineage of six to eight generations (7). Radiationinduced stem-cell apoptosis could result in irreversible intestinal tissue damage (8, 9). The number of crypts that survive after radiation damage determines how intact the intestinal mucosa is and, hence, how well an animal can survive the damage. Injury after prompt irradiation of hematopoietic and GI tissue is due to cell DNA damage, apoptosis and/or mitotic catastrophe occurring over a period of hours to days (10–12). The mechanisms of radiationinduced GI injury and mortality are not well understood and there are no FDA-approved pharmaceuticals to prevent or treat the acute radiation syndrome (5, 13–15). We recently demonstrated that natural delta-tocotrienol (DT3), an isomer of vitamin E, significantly enhanced survival in TBI mice. A single subcutaneous (s.c.) injection of DT3 (400 mg/kg) 24 h before TBI (8.75 Gy, c radiation, 0.6 Gy/min) protected CD2F1 mice from radiation-induced mortality with 100% 30-day survival, compared to 18% for vehicle-treated animals. DT3 protected mouse bone marrow and human hematopoietic CD34 þ cells from radiationinduced damage through Erk1/2 (extracellular signal-

We recently demonstrated that natural delta-tocotrienol (DT3) significantly enhanced survival in total-body irradiated (TBI) mice, and protected mouse bone marrow cells from radiation-induced damage through Erk activation-associated mTOR survival pathways. Here, we further evaluated the effects and mechanisms of DT3 on survival of radiationinduced mouse acute gastrointestinal syndrome. DT3 (75–100 mg/kg) or vehicle was administered as a single subcutaneous injection to CD2F1 mice 24 h before 10–12 Gy 60Co total-body irradiation at a dose rate of 0.6 Gy/min and survival was monitored. In a separate group of mice, jejunum sections were stained with hematoxylin and eosin and the surviving crypts in irradiated mice were counted. Apoptosis in intestinal epithelial cells was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and bacterial translocation from gut to heart, spleen and liver in irradiated mice were evaluated. DT3 (75 mg/kg) significantly enhanced survival in mice that received 10, 10.5, 11 or 12 Gy TBI. Administration of DT3 protected intestinal tissue, decreased apoptotic cells in jejunum and inhibited gut bacterial translocation in irradiated mice. Furthermore, DT3 significantly inhibited radiation-induced production of pro-inflammatory factors interleukin-1b and 6 and suppressed expression of protein tyrosine kinase 6 (PTK6), a stressinduced kinase that promotes apoptosis in mouse intestinal cells. Our data demonstrate that administration of DT3 protected mice from radiation-induced gastrointestinal system damage. Ó 2013 by Radiation Research Society

1These

two authors contributed equally to this work. for correspondence: Scientific Research Department, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Ave., Bethesda, MD 20889-5603; e-mail: [email protected]. 2Address

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regulated kinase 1/2) activation-associated mammalian target of rapamycin (mTOR) survival pathways (16). Eight distinct analogs of vitamin E have been designated: a-, b-, c-, d-tocopherol and a-, b-, c-, d-tocotrienol (17, 18). Previous studies had been conducted with tocopherols, the most commonly used vitamin E supplement and the most abundant vitamin E isoforms in human and animal tissue. During the last decade, tocotrienol research has gained substantial momentum. It has been shown that tocotrienols have beneficial effects on neuroprotection, anticancer, antioxidative stress, inhibition of nitric oxide (NO) and cholesterol levels–effects that are not exhibited by tocopherols (17–20). Furthermore, recent studies suggest that DT3 is the most effective anti-inflammatory and antioxidative stress agent among all the vitamin E forms tested in mouse macrophage and microglia cell lines (20, 21). In the current study, we further evaluated the effects and mechanisms of DT3 on mouse GI cell survival after 60Co c-photon irradiation using decreased dosages of DT3 (75–100 mg/ kg) (22). MATERIALS AND METHODS Mice Twelve- to 14-week-old CD2F1 male mice (Harlan Laboratories, Indianapolis, IN) were used according to methods described in previous reports (16). Mice were chosen randomly for each experimental group and housed in an AAALAC-approved facility at the Armed Forces Radiobiology Research Institute (AFRRI). The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC). Drug and Irradiation Delta-tocotrienol (DT3) was purchased from Yasoo Health Inc. (Johnson City, TN). The drug was solubilized in saline with 5% Tween-80 that also served as the vehicle for the animal studies. DT3 or vehicle was administered as a single s.c. injection to mice 24 h before TBI. Mice received 0 (sham), 10, 10.5, 11 or 12 Gy at a dose rate of 0.6 Gy/min in the AFRRI 60Co radiation facility. A DT3 dose of 75 mg/kg, determined to be one-quarter of the maximum tolerated dose (data not shown), was used in the survival study as described below. For other assays, a dose of 75–100 mg/kg DT3 (in 0.1 ml) was injected subcutaneous into mice weighing 23–30 g. Sham-irradiated mice were treated exactly the same as the gamma-irradiated animals except the 60Co source was not raised from the facility’s shielding water pool. After irradiation, mice were returned to their home cages. Survival Study The survival study consisted of two experimental groups (N ¼ 20 mice per group). Vehicle or 75 mg/kg DT3 in 0.1 ml was administered s.c. 24 h prior to TBI of mice weighing 25 6 2 g. The injection volume was adjusted for mice weighing greater than 28 g. Radiation doses for the vehicle control group were 10, 10.5 and 11 Gy, and the DT3-treated groups were given radiation doses 10, 10.5, 11 and 12 Gy at a dose rate of 0.6 Gy/min. Animals were irradiated in acrylic boxes (8 animals/box) and arranged in an array using plastic racks where they were confined for no more than 30 min. Mice were returned to their home cages at the end of the irradiation period. Survival was monitored for 30 days.

Histopathology of Jejunum Mouse jejuna obtained from mice 3.5 days postirradiation were fixed in Z-Fix (formaldehyde, methanol, ionized zinc buffer, Anatech Ltd., Battle Creek, MI) for at least 24 h. Samples were decalcified (Cal-EX for 3 h) and cross sections of jejunum were stained with hematoxylin and eosin (H&E). Jejunum sample slides were observed under the microscope for evaluating crypt and villi. A crypt microcolony survival assay was performed as described by Withers and Elkind (23). The circumference of a transverse cross section of the intestine was used as a unit. The number of surviving crypts of Lieberku¨hn (intestinal glands) was counted in each circumference to determine whether they contained at least 10 epithelial cells (either columnar enterocytes or goblet cells), a lumen and at least one Paneth cell (24). Six circumferences were scored per mouse and 6 mice were used to generate each data point. Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling (TUNEL) Assay The jejunum apoptotic cells were identified by TUNEL assay using a Cell Death Detection Kit (Roche Diagnostics Co., Mannheim, Germany) according to the manufacturer’s protocol. Briefly, jejunum sections obtained from mice at 12 h, 24 h and 3.5 days postirradiation were dewaxed at 608C for 30 min and washed in xylene. After dehydration in 100% ethanol, the jejunum sections were rehydrated by sequentially immersing the slides through gradient ethanol washes (95%, 90%, 80% and 70%) for 5 min each at room temperature. After washing in phosphate-buffered saline (PBS), sections were incubated with proteinase K working solution at 378C for 30 min and rinsed with PBS again. The cell nucleotides on the slides were labeled with fluorescein and were bound to the DNA 30-OH ends using terminal deoxynucleotidyl transferase. Slides were counterstained with 40,6diamidino-2-phenylindole (DAPI) mounting medium and a coverslip was applied. Fluorescence was observed under a Zeiss Axio Observer D1 fluorescence microscope. TUNEL-positive cells were scored as apoptotic cells. The numbers of apoptotic cells were counted in the high-power fields (HPF). Eight high-power fields were scored in each section, 6 sections per mouse and 6 mice were used to generate each data point. Evaluation of Gut Bacterial Translocation Mice euthanized in accordance with the IACUC protocol and the American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals (25) were dissected aseptically to isolate bacteria from selected tissues. Facultative bacteria were isolated from these tissues according to standard microbiological procedures in our laboratory (26). The apex of the heart was cut and the cut surface was immediately applied directly to surface of 5% sheep blood agar (SBA), colistin-nalidixic acid in 5% Sheep Blood Agar (CNA) and xylose-lysine-desoxycholate agar (XLD) media (BD Diagnostics, Sparks, MD). Liver and spleen samples were removed and homogenized by crushing with a sterile cotton or polyester swab in a sterile petri dish and inoculated immediately onto SBA, CAN and XLD media. Sheep blood agar and CNA were incubated in 5% CO2 at 358C for 18–24 h, and the XLD plates were incubated at 358C. Sheep blood agar is an enriched, nonselective medium, whereas CNA is selective for Gram-positive bacteria and XLD is selective for Gram-negative bacteria. Cultures without bacterial growth after 24 h were incubated for another 24 h. Individual colonies of isolated microorganisms were Gram-stained and subcultured on SBA to obtain a pure culture. They were incubated in 5% CO2 at 358C for 18–24 h and then observed to assure a pure culture, which was identified by a Vitek 2 Compact automated system (bioMe´rieux, Inc., Durham, NC) according to the manufacturer’s validated procedure.

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DT3 PROTECTS RADIATION-INDUCED GI INJURY

TABLE 1 Dose Response of DT3 for Radioprotection Postirradiation Survival time (day) 10

15

30

Radiation dose Gy

Vehicle

DT3

P value

10 10.5 11 12 10 10.5 11 12 10 10.5 11 12

80 65 75 * 30 0 5 * 30 0 0 *

100 100 85 75 100 100 70 35 100 95 60 10

NS ,0.009 NS

Survival (%)

,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001

Notes. CD2F1 male mice were injected s.c. with 75 mg/kg DT3 or vehicle and irradiated 24 h later with 10, 10.5, 11.0 or 12.0 Gy 60Co gamma radiation (N ¼ 20/group). Statistically significant differences between vehicle control and DT3-treated groups at each radiation dose and time point are given in the table. NS ¼ not significant. *Vehicle-treated group was not irradiated at 12 Gy because of the 5% and 0% survival observed in the 11 Gy vehicle-treated group, at 15 and 30 days after irradiation, respectively.

FIG. 1. DT3 protected mice against lethal gamma irradiation. DT3 (75 mg/kg) or vehicle was administered as a single subcutaneous (s.c.) injection to mice 24 h before total-body gamma-irradiation (TBI) at a dose rate of 0.6 Gy/min (N ¼ 20/group). Thirty-day survival was monitored after different doses of gamma radiation. There were significant increases in survival for all DT3-treated groups when compared to vehicle-treated control groups as shown in Table 1.

Immunoblotting

Statistical Analysis

Jejunum tissue was washed with PBS and immediately frozen in liquid nitrogen. The snap-frozen jejunum tissue was disrupted with mortar and pestle, resuspended in cold RIPA buffer (Sigma-Aldrich, St Louis, MO) containing a proteinase inhibitor cocktail and then homogenized. The protein concentration of the collected supernatant was determined using a bicinchoninic acid protein assay kit (BCA assay, Pierce, Rockford, IL). The proteins were diluted in 23 Laemmli sample buffer and denatured at 958C for 5 min. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were preblocked and probed with primary antibodies for PTK6 and b-actin as loading control (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA), according to the manufacturer’s instructions, followed by the appropriate horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Signal detection and analysis used an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL) and a Fuji Imaging System (27).

The Fisher’s exact test was used to compare survival among groups at the end of 10, 15 and 30 days. For other cell biology data, differences between means were compared by analysis of variance (ANOVA) and Student’s t tests. P , 0.05 was considered statistically significant. Results are presented as means 6 standard deviations of the mean as indicated.

Cytokine Quantification The snap-frozen jejunum tissues were disrupted with mortar and pestle, resuspended in cold PBS solution (pH 7.2) containing proteinase inhibitor cocktail and homogenized with Lysing Matrix D using FastPrep equipment (MP Biomedicals, LLC, OH), according to the manufacturer’s program. The whole lysate then was centrifuged at 12,000g for 15 min at 48C. The supernatant was collected and its total protein concentration was determined by use of a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Mouse ELISA (enzymelinked immuno sorbent assay) kits were purchased from R&D (Minneapolis, MN). Quantitative levels of cytokines in jejunum homogenate from samples obtained 12 h, 24 h and 3.5 days postirradiation were determined by ELISA assay following instructions provided by the manufacturers. The supernatant with an equivalent amount of protein from each sample was evaluated in duplicate. Statistical analysis was conducted from group samples of three mice.

RESULTS

Survival

A pilot study showed that the survival rates of mice that received DT3 at 75 mg/kg or 100 mg/kg were not significantly different 10 days after 10 Gy (100% survival at both drug doses) or 12 Gy (75% and 72% survival, respectively), (N ¼ 18/group). The total-body radiation doses used in this study included those associated with the standard end point for the GI syndrome (lethality within 10 days of exposure) (28). In addition, some radiation doses were associated with a combination of the GI and hematopoietic syndromes (e.g., lethality in 15 days postirradiation)(29, 30), while other doses were consistent with the end point for hematopoietic radiation damage (lethality at 30 days after irradiation) (28, 29). Therefore, in the present experiment, survival is reported at 10, 15 and 30 days after irradiation (Table 1 and Fig. 1), as has been done by other investigators (24, 29). DT3 (75 mg/kg) or vehicle was administered to mice 24 h before TBI at various radiation doses. There was significant (P , 0.009) protection from lethality by DT3 for mice receiving 10.5 Gy on day 10 postirradiation. Fifteen days after irradiation, survival for vehicle-treated animals

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(28). To evaluate the effects of DT3 on radiation-induced intestinal damage, jejunum was selected as the primary tissue and 3.5 days postirradiation was selected as the critical time point for histopathological analysis (31). Viable crypts of Lieberku¨hn were selected to study intestinal damage. Figure 2A shows the histopathology of hematoxylin and eosin-stained jejunum obtained from vehicle- and DT3-treated mice 3.5 days after irradiation. Jejunum samples from sham-irradiated mice served as controls. The specimens from unirradiated mouse jejunum exhibited healthy villi with palisades of epithelial cells and crypts extending into the depth of basal cell line. Radiation severely damaged mouse jejunum tissue, resulting in a significant decrease of viable crypts as well as villus length in 10 and 12 Gy irradiated groups and vehicle-treated mouse jejuna. The villi were collapsed and the epithelial cell cover vanished with the destroyed structure of the small intestine. In contrast, intestines from DT3-treated animals exhibited less structural injury than intestines from vehicle-treated mice in both the 10 and 12 Gy irradiated groups. Significant differences in viable circumferential crypt counts were observed in vehicle- vs. DT3-treated groups (N ¼ 6) on day 3.5 after 10 Gy (P , 0.001) and 12 Gy (P , 0.05), as shown in Fig. 2B. FIG. 2. DT3 administration increased survival of mouse intestinal crypts. Panel A: Histopathology of hematoxylin and eosin (H&E) stained jejunum obtained from vehicle- and DT3-treated mice 3.5 days after 0, 10 and 12 Gy irradiation. Sections of jejunum from representative mice in different groups are shown at 1003 magnification. Radiation severely damaged mouse jejunum tissue and resulted in a significant decrease of viable crypts and length of villi. DT3 administration increased survival of mouse intestinal crypts. Panel B: Viable crypt count in each section of jejunum from vehicle- and DT3treated groups on day 3.5 after 0, 10 and 12 Gy irradiation. *P , 0.05, **P , 0.01; N ¼ 6/group; mean 6 SD.

receiving 10, 10.5 and 11 Gy was 30%, 0% and 5%, respectively. This compared to a significant improvement in survival (P , 0.001) for DT3-treated animals that exhibited survival rates of 100%, 100% and 70%, for the same radiation doses, respectively. At 30 days postirradiation, DT3 survival was also significantly (P , 0.001) enhanced compared to vehicle-treated mice that received radiation doses of 10–11 Gy. Because there was 0% survival in the 11 Gy vehicle group, a vehicle control group for 12 Gy was not carried out in this experiment. If one assumes 0% survival for a 12 Gy vehicle group, then DT3 still provided significant (P , 0.05) protection on day 15 postirradiation for mice that received 12 Gy. DT3 Protected Mouse Gastrointestinal Epithelial Cells

The target organ of radiation-induced GI syndrome is the small bowel with the distinctive pathology being loss of the intestine’s epithelial lining (31). The classical histological end point in mice is the number of regenerating crypts measured in the small intestine at defined time 3.5 days

DT3 Inhibited Radiation-Induced Apoptosis in Mouse Jejunum

To evaluate the effect of DT3 on radiation-induced apoptosis in jejunum, mice were treated with DT3 or vehicle 24 h before exposure to 10 or 12 Gy radiation. Jejunum samples were collected from vehicle- or DT3-treated mice at 12 h, 24 h and 3.5 days after irradiation and apoptotic cells were identified by TUNEL assay. The numbers of TUNELpositive cells were counted in the high-power fields (HPF). Eight high-power fields were scored in each section, 6 sections per mouse and 6 mice were used to generate each data point. Figure 3A shows that very few spontaneous TUNEL-positive cells were observed in unirradiated cells. The number of TUNEL-positive cells increased and reached their highest levels at 12 h postirradiation in the vehicletreated group. DT3 treatment significantly protected jejunum cells from 10 and 12 Gy irradiation-induced apoptosis observed 12 h postirradiation. The numbers of TUNEL-positive cells were very low and there were no significant differences between vehicle- and DT3-treated mouse jejunum samples collected at 24 h (Fig. 3A) or 3.5 d (data not shown) after irradiation. Figure 3B shows TUNEL-positive cell counts from 6 mice per group in vehicle- vs. DT3-treated groups 12 and 24 h after 10 and 12 Gy. DT3 Protected GI Mucosal Barrier and Blocked Gut Bacterial Translocation in Irradiated Mice

High-dose irradiation degraded the GI mucosal and epithelial barrier and resulted in bacterial translocation from

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TABLE 2 DT3 Protected GI Mucosal Barrier and Blocked Intestinal Bacterial Translocation in Lethally Irradiated Mice Dose of radiation (Gy)

Treatment

Mice with bacterial growth/ total euthanized micea

10

Vehicle

4/5

12

DT3 Vehicle

0/6 3/3 (moribund)

DT3

2/5

Bacterial species isolatedb (number of mice) Streptococcus sanguinis (3*) Steptococcus uberis (1*) Sphingomonas paucimobilis (1**) NG (1) NG (6) Staphylococcus aureus (1*) Streptococcus mitis/ Streptococcus oralis (1*) Streptococcus sanguinis (1*) Sphingomonas paucimobilis (1**) Escherichia coli (1**) NG (3) Streptococcus sanguinis (1*) Enterococcus faecalis (1*) Escherichia coli (2**)

a

FIG. 3. DT3 protected against radiation-induced apoptosis in mouse jejunum cells. Panel A: Jejunum samples were collected from vehicle- or DT3-treated mice at 12 and 24 h after irradiation and apoptotic cells were identified by TUNEL assay. Slides were examined with a Zeiss fluorescence microscope (original magnification 1003). Immunofluorescence staining showed TUNEL (green) expression in representative samples from mice treated with vehicle or DT3 24 h before irradiation. DAPI (blue) defines cell nuclei. Panel B: TUNEL-positive cell counts per high-power field (HPF) from vehicleand DT3-treated groups at 12 and 24 h after 10 and 12 Gy irradiation. *P , 0.05, **P , 0.01; N ¼ 6/group; mean 6 SD.

the intestines into the blood that was detected in the liver, ventricular heart blood and spleen. Bacterial translocation was determined by bacterial isolation and identification. Results are presented in Table 2. In 10 Gy gamma-irradiated mice, bacterial culture was performed on day 11 postirradiation. No bacteria were isolated from mice treated with DT3 (0 of 6 mice). In contrast, bacteria were observed from 4 of 5 euthanized vehicle-treated mice (1 of 6 mice in this group died before culture was performed on day 11). Among these 4 mice, 1 had a polymicrobial infection by the Gram-positive coccus, Streptococcus sanguinis, and the Gram-negative rod, Sphingomonas paucimobilis. A single bacterial species was isolated from 3 of the 4 mice, Streptococcus sanguinis from 2 mice and Streptococcus uberis from 1 mouse. No

Bacterial species isolated from heart ventricular blood, liver and/or spleen in surviving DT3- or vehicle-treated septic mice on day 10 (12 Gy) or day 11 (10 Gy) after irradiation. N ¼ 6 mice/group except when mice died before day of bacterial culture. b NG ¼ no growth (no bacteria isolated); * ¼ Gram-positive; ** ¼ Gram-negative.

bacteria were isolated from the fifth vehicle-treated mouse on day 11. In 12 Gy gamma-irradiated mice, bacterial culture was performed on day 10 postirradiation. In DT3-treated mice, 1 of 6 mice died before culture on day 10. No bacteria were isolated from 3 of 5 DT3-treated mice. A single Gramnegative rod species, Escherichia coli, was isolated from 1 mouse and three species—one Gram-negative species, Escherichia coli, in combination with two Gram-positive species, Streptococcus sanguinis and Enterococcus faecalis—were found in another mouse, which demonstrated a polymicrobial infection in this mouse. In comparison, in the vehicle-treated group, 3 out of 6 mice died before culture on day 10 and bacteria were isolated from 3 moribund mice. Two of these mice had polymicrobial sepsis with two species, 1 with Gram-negative Sphingomonas paucimobilis and Gram-positive Streptococcus mitis/Streptococcus oralis and 1 with Gram-negative Escherichia coli and Grampositive Streptococcus sanguinis. A single species, Grampositive Staphylococcus aureus, was found in the third mouse.

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FIG. 4. DT3 inhibited radiation-induced production of IL-1b and IL-6 in mouse jejunum cells. Concentrations of IL-1b and IL-6 were evaluated by ELISA assay in homogenates of individual jejunum from vehicle- and DT3-treated mice at 12 h postirradiation. **P , 0.01; N ¼ 6/group; mean 6 SD.

DT3 Inhibited Radiation-Induced Synthesis of IL-1b and IL6 in Mouse Jejunum Cells

We next evaluated concentrations of selected proinflammatory factors in mouse jejunum cells at 12 h, 24 h and 3.5 days postirradiation because an acute inflammatory cytokine storm after ionizing radiation can cause mammalian cell damage (32). According to our previous studies (33, 34), pro-inflammatory cytokines (IL-1a, IL1b, IL-6 and IL-8) were evaluated by ELISA in jejunum tissue homogenates. An equal amount of total protein from whole jejunum tissue lysate of individual mice (1 mg total protein/mL of sample) was used to normalize results. Radiation-induced IL-1b and IL-6 expressions were observed in jejunum samples 12 h after irradiation, whereas levels of IL-1a and IL-8 in jejunum tissue lysate were undetectable using this assay. The cytokine detection limits were 1.8 pg/mL and 3.6 pg/mL for IL-1b and IL-6, respectively. Figure 4 shows that there were no significant differences in IL-1b and IL-6 production in the jejunum of untreated, vehicle-treated, or DT3-treated mice after shamirradiation (0 Gy). In contrast, a significant increase in IL1b and IL-6 were observed in untreated or vehicle-treated jejunum cells at 12 h after 10 and 12 Gy irradiation. DT3 suppressed the radiation-induced IL-1b increase in jejunum of mice after 10 and 12 Gy TBI (P , 0.01). Although IL-6 increased in DT3-treated and irradiated samples, the concentrations of IL-6 in DT3-treated samples were significantly lower than in vehicle-treated and non-treated groups (P , 0.01). At 24 h after irradiation, levels of IL-1b and IL-6 returned to baseline, as shown in nonirradiated cells and did not change thereafter (data not shown). DT3 Inhibits Protein Tyrosine Kinase 6 (PTK6) Activation in Mouse Intestinal Crypt Epithelial Cells

PTK6 is a stress-induced kinase that promotes apoptosis by inhibiting survival signaling in cells (35). Radiationinduced PTK6 expression in intestinal crypt cells has been reported to be correlated with DNA damage-mediated apoptosis in the small intestine (36). We asked whether the radioprotective effects of DT3 are mediated through the

FIG. 5. DT3 inhibited protein tyrosine kinase 6 (PTK6) activation in mouse intestinal cells. PTK6 activation was evaluated by Western blotting in mouse intestinal crypt epithelial cells. Mouse jejunum samples were collected from vehicle- or DT3-treated mice 12 h after sham (0 Gy), 10 Gy (panel A) or 12 Gy (panel B) irradiation. Western blots from 3 mouse samples per group demonstrate representative PTK6 expression in DT3- and vehicle-treated irradiated mice. Betaactin served as loading control. (N ¼ 6/group).

PTK6 signal pathway. Mouse jejunum samples were collected from vehicle- and DT3-treated mice 12 h after sham-irradiation, 10 and 12 Gy irradiation. Individual jejunum samples from each group were evaluated (N ¼ 6). Figure 5 shows results from an immunoblotting assay. PTK6 protein was not detectable in sham-irradiated intestine cells from either vehicle-treated or DT3-treated mice. In contrast, PTK6 expression was observed in 2 out of 3 samples evaluated 12 h after 10 Gy and 3 of 3 samples evaluated 12 h after 12 Gy irradiation. Administration of DT3 24 h before irradiation inhibited PTK6 production in jejunum after 10 and 12 Gy TBI. PTK6 production was not observed at 24 h or on day 3.5 postirradiation (data not shown). DISCUSSION

Our previous study demonstrated that natural DT3 (400 mg/kg) from palm oil or rice bran oil protected mice from 8.75 Gy TBI and resulted in 100% 30-day survival compared with 18% survival in vehicle-treated control mice (16). DT3 as a pro-proliferative and anti-apoptotic agent inhibits radiation-induced DNA damage in hematopoietic stem and progenitor cells and protects mouse and human hematopoietic stem and progenitor CD34 þ cells from radiation-induced mortality through increased bone marrow regeneration capacity and hematopoietic stem and progenitor cell proliferation(37). DT3 administered either 24 h before or 6 h after irradiation increased mouse bone marrow myeloid cell numbers. The mechanism of DT3-mediated radioprotection of hematopoietic cells has been attributed to DT3’s stimulation of Erk activation-associated mTOR survival pathways (16). Satyamitra and colleagues (22, 38) reported the prophylactic and therapeutic efficacy of DT3 administered by subcutaneous injection at doses of 75,

DT3 PROTECTS RADIATION-INDUCED GI INJURY

150 or 300 mg/kg in 9.25 Gy gamma-irradiated mice. Their data demonstrated that DT3 protected the mouse hematopoietic system as reflected in higher levels of white blood cells, neutrophils, reticulocytes, platelets and lymphocytes. In addition, their drug safety data showed no changes in body weight or mortality in mice administered DT3 s.c. up to 1,000 mg/kg. In the current study we determined that DT3 (75 mg/kg) protected CD2F1 male mice from lethal radiation doses (10.5–12 Gy) as shown by survival at 10, 15 and 30 days postirradiation. The hematopoietic and GI systems are among the most radiation-sensitive organs (1). The acute generalized GI syndrome appears after high-dose TBI, which results in the death of mice within 10 days (24, 31, 39). Death approximately 10 days after irradiation in the CD2F1 mouse is generally indicative of animals succumbing to the initial phases of the GI syndrome (24). However, an accelerated bone marrow syndrome can also cause death in mice (40). The hematopoietic and GI syndromes are on a continuum with a gradual transition from one syndrome to the other with increasing doses of radiation (2, 24). Results from our study demonstrated a significant radioprotective effect on survival for DT3 10 days after 10.5 Gy, as well as 15 and 30 days postirradiation (10–12 Gy). These results suggest that the radiation doses used in the current study resulted in a combination of hematopoietic and GI injury and demonstrates that DT3 at relatively low doses (75 mg/ kg) is protective against both radiation-induced hematopoietic and GI syndromes. We further evaluated the radioprotective effects of DT3 on the mouse GI system after lethal doses of radiation. Radiation induces (1) loss of intestinal crypts and damage of crypt stem cells, (2) depletion of epithelial cell population and denudation of the intestinal wall, and (3) breakdown of the GI mucosal barrier (31). The GI tract is the source of translocation of bacteria and sepsis after radiation-induced injury to the intestinal lining (41, 42). Our data demonstrate that a single administration of DT3 24 h before 10 or 12 Gy irradiation protected mouse intestinal tissue from radiation damage, inhibited jejunum cell apoptosis, and enhanced survival of intestinal crypts after irradiation. In addition, DT3 blocked bacterial translocation and sepsis in irradiated mice. Sepsis was demonstrated in 7 of 8 vehicle-treated mice that received 10 or 12 Gy. In contrast, sepsis was detected in only 2 of 11 DT3-treated irradiated mice, and these 2 mice were irradiated with 12 Gy. Both Gramnegative and Gram-positive bacterial species were isolated from vehicle- and DT3-treated tissues, suggesting that DT3 did not selectively protect mice from bacterial infection, but may have prevented bacterial translocation from the GI tract in lethally irradiated mice. Although the radiation-induced acute GI syndrome has been studied widely, the mechanisms underlying intestinal tissue damage from radiation are still not completely understood (13). An acute inflammatory cytokine storm after ionizing irradiation results in mammalian cell damage (32). Local and systemic inflammation can alter the long-

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term state of immunity, exacerbating autoimmune disorders that can lead to multiple organ failure. We recently reported that radiation-induced inflammatory cytokine release was associated with osteoblastic cell senescence (33). Furthermore, IL-1 can sensitize mice to both early and late morbidity and mortality from total abdominal irradiation. Therefore, blocking IL-1 production may inhibit the development of radiation-induced GI injury (43). To mitigate this side effect of radiotherapy, approaches that focus on cytokine neutralization have been developed to maintain control of the inflammatory response (44). In this study, we found that radiation enhanced production of proinflammatory cytokines IL-1b and IL-6 in mouse intestinal cells, and that increases in levels of these cytokines occurred in a radiation dose-dependent manner. Administration of DT3 before TBI significantly inhibited radiation-induced production of IL-1b and IL-6 in mouse jejunum. This reduced production of pro-inflammatory cytokines after DT3 administration may contribute to protecting mouse intestinal epithelial cells from radiation-induced inflammation. Our data are consistent with recent reports that demonstrated the anti-inflammatory effects of DT3 in BV2 microglia (20) and macrophage cells (21) in in vitro studies. In this study we found that levels of IL-6 in vehicletreated and irradiated jejunum samples were lower than in untreated and irradiated samples. The reason for this phenomenon is currently not known and warrants further investigation. One possibility is that an injection site reaction stimulated the immune system. Nevertheless, DT3 significantly decreased IL-6 in irradiated jejunum samples compared to vehicle-treated groups. Results from our study demonstrate that radiation-induced apoptotic (TUNEL-positive) jejunum cells increased at 12 h postirradiation, which may cause an irreversible decrease in number of intestinal crypts (8, 9). This enterocyte depletion can eventually result in mucosal barrier breakdown, bacterial translocation into the blood stream, and lethality of TBI mice (28). DT3 inhibited PTK6 activation and suppressed apoptosis in mouse intestinal crypt epithelial cells. PTK6 (also called BRK or Sik) is an intracellular tyrosine kinase that is related to members of the Src family and contains SH3 and SH2 domains as well as a carboxyl regulatory tyrosine (45). PTK6 is expressed in epithelial cells of the GI tract, prostate, mouth and skin, as well as lymphocytes (45–47). After DNA damage, induction of PTK6 is required for efficient apoptosis and inhibition of prosurvival factors AKT and Erk 1/2 (36). Haegebarth et al. (36) reported radiation-induced PTK6 expression in crypt epithelial cells of mouse small intestine, and this induction of PTK6 corresponded with DNA damage-induced apoptosis (36). Our previous study demonstrated that the radioprotective effects of DT3 on mouse and human hematopoietic cells were mediated through Erk 1/2 signaling (16). Here we further demonstrated that DT3 suppressed the radiation-induced PTK6 in mouse intestinal cells, suggesting that the mechanisms of DT3 effects on

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survival of radiation-injured mouse hematopoietic cells and intestinal epithelial cells may be through regulation of Erk and PTK6 signal transduction pathways. However, DT3induced activation of Erk and survival factor AKT in irradiated mouse intestinal tissue was not observed in this study. In conclusion, our data indicate that DT3, as an antiapoptotic agent, inhibited pro-inflammatory cytokine production and PTK6 expression in mouse intestinal tissue after exposure to c radiation and protected mice from lethaldose, radiation-induced acute GI syndrome. This isomer of vitamin E may have applications in protecting against radiation injury from emerging radiological and nuclear threats and radiotherapy-induced side effects to normal tissue.

12.

13. 14.

15. 16.

17.

ACKNOWLEDGMENTS The views expressed here do not necessarily represent those of the Armed Forces Radiobiology Research Institute, the Uniformed Services University of the Health Sciences, or the Department of Defense. The authors would like to thank Dr. Kushal Chakraborty, Mr. Kevin Hieber and Ms. Roli Pessu for technical assistance. This study was supported by Armed Forces Radiobiology Research Institute intramural grants (RAB2EI and RAB2GO) to MX and RAA610 to SPG. Received: May 2, 2013; accepted: August 29, 2013; published online: November 26, 2013

18. 19.

20.

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