Selenomethionine protects against adverse biological effects induced by space radiation

Free Radical Biology & Medicine, Vol. 36, No. 2, pp. 259 – 266, 2004 Copyright D 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter

doi:10.1016/j.freeadbiomed.2003.10.010

Original Contribution SELENOMETHIONINE PROTECTS AGAINST ADVERSE BIOLOGICAL EFFECTS INDUCED BY SPACE RADIATION $ ANN R. KENNEDY, * JEFFREY H. WARE,* JUN GUAN, * JEREMIAH J. DONAHUE, * JOHN E. BIAGLOW, * ZHAOZONG ZHOU, * JELENA STEWART, * MARCELO VAZQUEZ, y and X. STEVEN WAN* *Department of Radiation Oncology, University of Pennsylvania School of Medicine, 195 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA, USA; and y Brookhaven National Laboratory, Upton, NY, USA (Received 21 August 2003; Revised 8 October 2003; Accepted 15 October 2003)

Abstract—Ionizing radiation-induced adverse biological effects impose serious challenges to astronauts during extended space travel. Of particular concern is the radiation from highly energetic, heavy, charged particles known as HZE particles. The objective of the present study was to characterize HZE particle radiation-induced adverse biological effects and evaluate the effect of D-selenomethionine (SeM) on the HZE particle radiation-induced adverse biological effects. The results showed that HZE particle radiation can increase oxidative stress, cytotoxicity, and cell transformation in vitro, and decrease the total antioxidant status in irradiated Sprague – Dawley rats. These adverse biological effects were all preventable by treatment with SeM, suggesting that SeM is potentially useful as a countermeasure against space radiationinduced adverse effects. Treatment with SeM was shown to enhance ATR and CHK2 gene expression in cultured human thyroid epithelial cells. As ionizing radiation is known to result in DNA damage and both ATR and CHK2 gene products are involved in DNA damage, it is possible that SeM may prevent HZE particle radiation-induced adverse biological effects by enhancing the DNA repair machinery in irradiated cells. D 2003 Elsevier Inc. All rights reserved. Keywords—HZE particles, Selenomethionine, Free radicals, Oxidative stress

biological effects of HZE particle radiation. Knowledge in both of these areas is needed for the development of effective countermeasures for long-term space travel in the future. By definition, all types of ionizing radiation generate ions, which can lead to the formation of free radicals and reactive oxygen species. Thus, ionizing radiation is a pro-oxidant. Oxidative stress is a term that describes a state of imbalance between the pro-oxidants and antioxidants, favoring the pro-oxidants [1]. Increased oxidative stress has been demonstrated after radiation exposure in various in vitro and in vivo model systems [2– 4] and is known to be associated with space flight [5]. Because oxidative stress has been implicated in various human degenerative diseases, such as atherosclerosis [6,7], cancer [6,8 – 10], Parkinson’s disease [11,12], and Alzheimer’s disease [13,14], it is probable that some of the adverse biological effects induced by space radiation are related to radiation-induced oxidative stress. In the present study, beams of iron ions generated by the Alternating Gradient Synchrotron (AGS) at the

INTRODUCTION

Exposure to ionizing radiation during space travel is expected to result in an increased risk of cancer and other adverse biological effects in astronauts. One type of radiation of particular concern for astronauts during space travel comes from highly energetic, heavy, charged particles known as HZE particles. Relatively little is known about the biological effects induced by radiation with HZE particles, as this type of radiation is not normally encountered on Earth and only a few sources of HZE particles are available in the world for experimental studies. Even scarcer is information about potential countermeasures capable of protecting against the $

This work was supported by grants from the National Space Biomedical Research Institute (Project RE00204) and from NASA (02-OBPR-02-0019-0039). Address correspondence to: Ann R. Kennedy, 195 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6072. Fax: (215) 898-0090, E-mail: [email protected]. 259

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Brookhaven National Laboratory (BNL) were used as sources of HZE particles to study adverse biological effects induced by space radiation in cultured human cells and Sprague – Dawley rats. The protective effects of SeM, a potential cancer preventive agent with demonstrated anticarcinogenic activities in animal studies [15 – 18] and human cancer prevention intervention trials [19,20], on the adverse biological effects of HZE particle radiation were also evaluated.

MATERIALS AND METHODS

Cells and cell culture Human breast epithelial (MCF10) cells [21] were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 (F12) medium supplemented with 5% horse serum (Sigma), 20 ng/ml epidermal growth factor, 0.1 Ag/ml cholera toxin, 10 Ag/ml insulin, and 0.5 Ag/ml hydrocortisone. Human thyroid epithelial (HTori3) cells [22] were cultured in DMEM supplemented with 7% fetal bovine serum. The cells were subcultured by treatment with trypsin – ethylenediaminetetraacetic acid (EDTA) whenever the cells become confluent. Dichlorofluorescein (DCF) fluorometric assay The DCF assay was performed as previously described elsewhere [23]. Briefly, confluent MCF10 cells were incubated in medium containing 0 (control), 10, or 20 AM SeM for 16 h, washed with PBS, and further incubated with 50 AM 2V,7V-dichlorofluorescein acetate (DCFH-DA) substrate solution for 30 min. After incubation, the cells were washed with PBS and irradiated with 1 or 5 GeV iron ions at doses of 0 (sham radiation), 5, 10, 20, 40, or 80 cGy while covered with 200 Al of PBS with or without SeM in PBS. Immediately following the radiation exposure, the plates were read three times at 90-s intervals with a Bio-Tek FLx800 fluorescence plate reader at excitation and emission wavelengths of 485 and 528 nm, respectively. The level of fluorescence in the sham-radiation treatment group was subtracted from the fluorescence readings for the radiation treatment groups to calculate the increase in cellular fluorescence. Cell survival assay MCF10 cells were pretreated with 5 AM SeM for 18 h in medium and irradiated with 5 GeV iron ions at specified doses. After the radiation exposure, the cells were dissociated by treatment with trypsin – EDTA, resuspended in medium with or without 5 AM SeM, plated in T-25 tissue culture flasks at 300 to 450 cells per flask, and cultured for 6 days. At the end of the incubation period, the cell colonies were fixed and stained with

crystal violet and methylene blue dissolved in 90% ethanol and counted under a dissection microscope. The number of surviving cell colonies was divided by the number of cells plated to calculate the clonogenic survival for each flask. This is known as a clonogenic survival assay, as it involves a determination of the number of cells that survived the radiation exposure and were capable of forming colonies containing greater than 50 cells, called a cell clone. The surviving fraction data were plotted against the radiation doses to calculate radiation sensitivity constants according to the multitarget theory [24] using the equation S = nekD, where S is the surviving fraction, n represents the number of targets, k is the radiation sensitivity constant, and D is the dose of radiation (cGy). Animal care and serum total antioxidant status assay Female Sprague – Dawley rats approximately 5 weeks old were fed AIN-93G diet with or without supplementation with SeM (12 Ag of SeM/g of diet) for 3 days, then irradiated with 200 cGy of 1 or 5 GeV iron ions. Five rats were used in each treatment group at each dose level. The animals were killed 4 h after the radiation exposure by carbon dioxide inhalation. Blood was collected by cardiopuncture and kept at room temperature for 30 min for clot formation. After a 2-min centrifugation at 10,000 rpm to separate the serum from the blood clot, clear sera were collected into microcentrifuge tubes and stored at 70jC before analysis. The effect of HZE particle radiation on oxidative stress in Sprague – Dawley rats was assessed by measuring the total antioxidant status in the blood by a colorimetric assay system developed by Randox Laboratories Ltd. (Antrim, UK), which is currently being used for analyses of total antioxidant status in blood samples from astronauts [25]. This assay is based on the principle that 2, 2V-azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS), when incubated with a peroxidase (metmyoglobin) and H2O2, produces ABST*+ radicals with a relatively stable blue-green color that can be measured at a wavelength of 600 nm. In the presence of antioxidants, the formation of colored ABTS*+ radicals is suppressed and the magnitude of suppression is proportional to the antioxidant concentration in the reaction solution. Thus, this assay system measures the combined power of antioxidants which is collectively referred to as total antioxidant status. To accommodate the need for simultaneous measurement of multiple samples, the assay method described in the manufacturer’s instructions was modified into a 96-well plate format with wells on each row assigned to reagent blank, standard, or test samples, respectively. To perform the assay, 4 Al of double-distilled water, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (HTCA) standard solution, or a serum/plasma sample was mixed with 200 Al chromogen solution (6.1

Selenium and space radiation

AM metmyoglobin and 610 AM ABTS) in each well, and the plate was placed into a PowerWave340 Microplate Spectrophotometer (Bio-Tek Instruments, Inc.) for a minimum of 15 min, with temperature maintained at 37jC to allow the temperature to reach equilibrium. To initiate the reaction, 4 Al of diluted substrate (100 AM H2O2 in stabilized form) was applied in each well, and the plates were shaken for 3 s and read at a wavelength of 600 nm four times at 1-min intervals. The total antioxidant status in the samples was calculated based on the slopes of the linear kinetic lines determined in the wells as follows and expressed in units of millimolar HTCA-equivalent per milliliter of serum or plasma: Total antioxidant status ðmMÞ ¼

slope for blank  slope for sample slope for blank  slope for standard concentration of standard:

Soft agar colony formation assay The malignant transformation of human thyroid epithelial (HTori-3) cells [22] was quantitated by a soft agar colony formation assay that measures the ability of the cells to grow under anchorage-independent conditions. HTori-3 cells have previously been adapted for studies of radiation transformation [26,27]. Anchorage-independent growth is a phenotypic change associated with the ability of cells to form tumors in animals; and tumor formation has previously been reported within 7– 20 weeks after irradiated HTori-3 cells are transplanted into athymic nude mice [26]. To carry out the experiments, HTori-3 cells were pretreated with 5 AM SeM for 18 h in medium and irradiated with 5 GeV iron ions at 125 cGy. Two weeks after the radiation exposure, the cells were dissociated by treatment with trypsin –EDTA, plated in 24-well tissue culture plates at 2000 cells per well, and cultured for 3 weeks. At the end of the incubation period, the cell colonies were stained with neutral red and counted under a dissection micro- scope to calculate the yield of radiation-induced transformants. Real-time polymerase chain reaction (PCR) Cultured HTori-3 cells were incubated in control medium or medium supplemented with 5 AM SeM for 24 h and irradiated with 5 GeV iron ions at a doses of 0 (sham radiation control) or 40 cGy. After the radiation exposure, the cells were incubated for an additional 12 h, then lysed for RNA isolation using a total RNA isolation kit from Ambion (used according to the manufacturer’s instructions). cDNA was synthesized via reverse transcription using SuperScript II kit and oligo(dT)12 – 18 as a primer (both from Invitrogen). The ATR (A) and CHK2 (B) transcripts were quantified by real-time PCR using ATR forward primer, 5VTGC AGA GTG CCA GGG TAG

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CT 3V; ATR reverse primer, 5VTGG TGA ACA TCA CCC TTG GA 3V; ATR probe, 5VFAM-TCA CCA CCA GAC AGC CTA CAA TGC TCT C-TAMRA3V; CHK2 forward primer, 5VGGA GAG CTG TTT GAC AAA GTG GT 3V; CHK2 reverse primer, 5V TCT GGC TTT AAG TCA CGG TGT ATA A 3V; and CHK2 probe, 5VFAM-CCA GAT GCT CTT GGC TGT GCA GTA CCT-TAMRA3V. Targets were amplified using AmpliTaq Gold (Roche) in Buffer II and 5 mM MgCl2, through 40 two-step cycles (95jC for 15 s and 60jC for 1 min) in an ABI PRISM 7700 Sequence Detection System. Standard curves were generated for each target and used for target quantity determination in the unknown samples (in triplicates). ATR and CHK2 mRNA levels were normalized to the quantity of GAPDH mRNA. Data and statistical analysis Means and standard deviations were calculated and presented for all data points that had at least three measurements. The mean values of total antioxidant status, colony formation efficiency, and mRNA abundance were compared between treatment and control groups by Student’s t test. The dose – response curves were established by linear or semilogarithm regression analyses using radiation doses as the independent variables and the responses (increase in fluorescence or cell surviving fraction) as the dependent variables. Statistical analyses were performed using Prism Version 2.0 statistical software (GraphPad Software, San Diego, CA, USA). RESULTS

In the present study, the adverse biological effects induced by HZE particle radiation were characterized in terms of the levels of oxidative stress and cell survival in irradiated MCF10 cell cultures, the yields of malignantly transformed cells in irradiated HTori-3 cell cultures, and total antioxidant status in irradiated Sprague – Dawley rats. Because HZE particle radiation is expected to result in DNA damage in the irradiated cells, the expression levels of two genes, ATR and CHK2, were also measured in the cells after the radiation exposure. The levels of oxidative stress in irradiated MCF10 cell cultures with or with SeM supplementation were determined by the DCF assay. The results demonstrated that radiation with 1 and 5 GeV iron ions in the dose range 5 to 80 cGy increased the levels of oxidative stress in irradiated MCF10 cell cultures in a highly dose-dependent manner (Fig. 1A). Addition of SeM to the cell culture medium at nontoxic concentrations (10 – 20 AM) completely prevented the radiation-induced increase in oxidative stress level (Fig. 1A). The exposure to HZE particle radiation also resulted in a dose-dependent decrease in clonogenic survival of

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5-GeV iron ions

1-GeV iron ions

***

110

100

Radiation + SeM

SeM

Radiation

Control

Radiation + SeM

**

SeM

80

Radiation

*

90

Control

Total Antioxidant Status (% of Control)

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Fig. 2. Effect of dietary supplementation with SeM on HZE particle radiation induced a decrease in total antioxidant status in Sprague – Dawley rats. Five rats were used in each treatment group at each dose level and the results are presented as means F SE. The level of total antioxidant status in rats fed the control diet decreased significantly after exposure to radiation with 1 GeV (*p < .05) or 5 GeV (**p < .01) iron ions. The total antioxidant status in rats fed the diet supplemented with SeM was higher than that of the sham radiation control groups with or without the radiation exposure; the increase was statistically significant (***p < .001) in the rats fed the SeM-supplemented diet and irradiated with 5 GeV iron ions.

Fig. 1. Effect of SeM on HZE particle radiation-induced oxidative stress (A) and cytotoxicity (B) in MCF10 cells. (A) The experiments were carried out using at least six replicates for each treatment group at each dose level and the results are presented as means F SD. (B) Two separate experiments were performed, with four flasks per point in each experiment. Each data point represents the average (mean F SD) surviving fraction values from the four flasks in one experiment. Significant protection against HZE particle radiation-induced oxidative stress and cytotoxicity by SeM supplementation was observed.

MCF10 cells irradiated with 5 GeV iron ions (Fig. 1B). The radiation sensitivity constants for MCF10 cells irradiated with or without concurrent treatment with SeM were 0.0330 and 0.0147, respectively. The doses of radiation required to yield 37% cell survival (known as the D0 or the D37, which equals 1/ –k) were 30.3 and 68.0 cGy, respectively, for the cells irradiated without and with the additional SeM treatment. These results suggest that treatment with SeM protected MCF10 cells from 5 GeV iron ion radiation-induced cell killing by a factor of 2.24. The total antioxidant status was measured in Sprague – Dawley rats exposed to radiation with 1 or 5 GeV iron

Fig. 3. Effects of SeM on HZE particle radiation-induced transformation of HTori-3 cells. Two separate experiments were performed; results from these experiments exhibited similar trends for the data. Results from a representative experiment are shown. Each data point represents the average (mean F SD) of four measurements. The sham-irradiated cells were included in the experiments as controls. The results indicated that the soft agar colony formation efficiency for HTori-3 cells increased from 1.51% (in the sham radiation control) to 13.85% after the radiation exposure and the increase was statistically significant (***p < .0001). Soft agar colony formation efficiencies for HTori-3 cells treated with SeM alone or with radiation were 0.54 and 0.35%, respectively, which were both below the soft agar colony formation efficiency of the control group (**p < .01).

Selenium and space radiation

ions. The results demonstrated that total antioxidant status in serum decreased by approximately 15% after radiation exposure (Fig. 2) and the decrease was statistically significant ( p < .0120 for 1 GeV and p < .0035 for 5-GeV). The decrease in total antioxidant status associated with the exposure to HZE particle radiation was completely prevented by supplementation of the rat diet with a nontoxic level of SeM (Fig. 2). The effect of HZE particle radiation on HTori-3 cell transformation was determined by measuring the yield of colonies growing under anchorage-independent conditions after exposure to radiation with 5 GeV iron ions.

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The results demonstrated a more than 8-fold increase in soft agar colony formation efficiency in irradiated HTori-3 cell cultures (Fig. 3, p < .005), suggesting that HZE particle radiation is highly effective in inducing the malignant transformation of HTori-3 cells in vitro. The soft agar colony formation efficiencies of HTori-3 cells cultured in medium containing 5 AM SeM with or without exposure to the HZE particle radiation were both below the soft agar colony formation efficiency of the shamirradiated HTori-3 cells cultured in medium without SeM ( p < .005). These results indicate that treatment with SeM not only completely prevented the 5 GeV iron ion radiation-induced transformation of HTori-3 cells, but also reduced the baseline level of transformed HTori-3 cells. The effect of HZE particle radiation on ATR and CHK2 gene expression in cells was evaluated by a real-time PCR technique in HTori-3 cells cultured in medium with or without supplementation with 5 AM SeM. The results indicated that treatment with 5 AM SeM significantly increased the level of ATR mRNA in HTori3 cells with and without the radiation exposure (Fig. 4A). Compared with the level of ATR mRNA in shamirradiated HTori-3 cells cultured in control medium, the level of ATR mRNA was 42% higher in sham-irradiated HTori-3 cells cultured in SeM supplemented medium and 94% higher in HZE particle-irradiated HTori-3 cells cultured in SeM-supplemented medium. Treatment with 5 AM SeM also significantly increased the level of CHK2 mRNA in the irradiated HTori-3 cells by 99% (Fig. 4B). DISCUSSION

Fig. 4. Effects of SeM on ATR and CHK2 gene expression in HTori-3 cells irradiated with 5 GeV iron ions. ATR and CHK2 mRNA levels determined by the real-time PCR method were normalized to the quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The level of ATR mRNA in HTori-3 cells cultured in SeM supplemented medium was significantly higher than that of the cells cultured in control medium (*p < .05). The levels of ATR and CHK2 mRNA in the cells irradiated in the presence of SeM were also significantly higher than those in the cells irradiated without SeM in medium (**p = .01 for ATR, ***p = .0001 for CHK2).

The present study was undertaken to characterize HZE particle radiation-induced adverse biological effects and evaluate the effect of SeM, a potential cancer chemopreventive agent, on HZE particle radiation-induced adverse biological effects. The results demonstrate that HZE particle radiation resulted in increased oxidative stress, increased cell transformation and decreased cell survival in vitro, and decreased total antioxidant status in irradiated Sprague – Dawley rats. The results also show that these adverse biological effects of HZE particle radiation were preventable by treatment with SeM, an organic selenium compound with demonstrated anticarcinogenic activities in animals treated with selenium at levels higher than those required for nutritional needs [15 – 18] and in several human cancer prevention intervention trials [e.g., 19,20]. The observed protection by SeM against HZE particle radiation induced cell killing is an important finding as it could challenge the current radiobiological concept that high LET radiationinduced lethal damage cannot be modified. The decrease in total antioxidant status observed in the irradiated rats suggests that HZE particle radiation also induced oxida-

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tive stress in vivo. These results are consistent with the previously observed reduction in total antioxidant status after long-duration spaceflights [25]. The protective effect of SeM on the HZE particle radiation-induced decrease in total antioxidant status in rats is also consistent with the observed protective effects of SeM against HZE particle radiation-induced oxidative stress and cytotoxicity in MCF10 cells and malignant transformation in HTori-3 cells. SeM is the form of selenium that has been chosen by the National Cancer Institute (NCI) for current studies of selenium as a cancer preventive agent. The largest intervention study using SeM as the cancer preventive agent is a Phase III prostate cancer prevention trial known as the Selenium and Vitamin E Cancer Prevention Trial [28], with SeM being administered to trial participants at a dose of 200 Ag/d. Selenium has a relatively long half-life in mammals, and chronic exposure to high doses of selenium is known to be associated with growth failure and weight loss, as well as liver damage, splenomegaly, pancreatic enlargement, anemia, dermatitis, hair loss, and abnormal nails [29]. In previously documented rat studies, daily doses of 0.75 and 1.5 mg SeM/kg body wt per day for 28 days were considered to be nontoxic doses [30]. In the present study, the animal experiment was performed with a SeM dose of 1 mg/kg body wt per day for 3 days. This relatively high dose was selected to compensate for the short treatment period and to avoid the possibility of experimental failure due to insufficient dose of the supplement agent while minimizing the treatment groups in the experiment. No toxic effects of SeM treatment were observed in this study. The fact that SeM at a nontoxic dose prevented all HZE particle radiation-induced adverse biological effects observed in the present study suggests that SeM has the potential to be an effective countermeasure against space radiationinduced adverse biological effects. The mechanism for the cancer preventive activity of selenium is not clear and can only be speculated at this time. One hypothesis attributes the cancer preventive activity of selenium to its essential role in maintaining the activities of the antioxidant enzymes glutathione peroxidase and thioredoxin reductase [31,32]. It is well established that the exposure of cells and organisms to ionizing radiation produces many different types of molecular damage. Ionizing radiations can interact with cells through either direct or indirect actions; the former refers to the direct interactions of radiation with critical targets in the cell, whereas the latter refers to the interactions of radiation with other molecules (primarily water) or atoms in or around the cells, resulting in the production of free radicals capable of damaging critical targets in cells. Indirect actions are associated primarily with low-LET radiations (e.g., x and g rays), while direct actions

predominate for high-LET radiations, such as that arising from HZE particles. The indirect effects involving free radicals can be modified or prevented by a variety of antioxidant enzymes or small-molecular-weight free radical scavengers. However, antioxidant enzymes are not expected to affect biological effects brought about by the direct actions of high-LET radiations, as recently reviewed [33]. Thus, the radioprotective effects of SeM against HZE particle radiation-induced adverse biological effects may not entirely be attributable to the essential role of selenium in maintaining antioxidant enzyme activities. We hypothesize that SeM may serve as a countermeasure for HZE particle radiation-induced adverse biological effects through its ability to regulate the expression of genes involved in the repair of radiation-induced DNA damage. It is conceivable that the ultimate biological consequence of DNA damage caused by HZE particle radiation is critically dependent on whether the damage is promptly and properly repaired. A previously observed effect of SeM in activating the p53 tumor suppressor protein by a redox mechanism suggests that the p53 pathway of DNA repair machinery is activated in SeMtreated cells [34]. In one of our ongoing microarray studies using the LNCaP cell line (a human prostate carcinoma cell line), treatment with SeM was shown to upregulate ATR and CHK2 gene expression; both of these genes are important components of the DNA damage response pathway [35,36]. ATR is one of the central components of the DNA damage response pathway [35] whereas CHK2 is a cell cycle checkpoint regulator and putative tumor suppressor [36,37]. Lack of functional ATR has been linked to increased sensitivity of human fibroblasts to ionizing radiation and defects in cell cycle checkpoints [38]. Defects in CHK2 are believed to contribute to the development of both hereditary and sporadic human cancers [39]. The results of the present study suggest that supplementation with SeM might prevent the malignant transformation of cells by allowing the checkpoint genes ATR and CHK2 to be expressed at higher levels following HZE particle radiation exposure. Upregulation of ATR and CHK2 gene expression may allow the cells to handle HZE particle-induced DNA damage in a particularly efficient manner, resulting in protection against the adverse biological effects associated with DNA damage caused by HZE particle radiation. It is conceivable that increased expression of ATR and CHK2 will keep cells from going through mitosis until the damage is repaired, thereby preventing the damage from being fixed such that mutations and malignant transformation can occur. It needs to be pointed out that the effects of SeM treatment on ATR and CHK2 gene expression were demonstrated only at the mRNA level in the present study. In the absence of evidence showing

Selenium and space radiation

that the protein products of these genes were also increased after SeM treatment, the hypothesized involvement of ATR and CHK2 genes in SeM-mediated protection against HZE particle radiation-induced adverse biological effects remains to be proven. In summary, our results indicate that exposure to HZE particle radiation results in increased oxidative stress, cell killing, and malignant transformation in cultured human epithelial cells and decreased total antioxidant status in Sprague –Dawley rats, and that these adverse biological effects can be effectively prevented or suppressed by nontoxic doses of SeM at the time of radiation exposure. These findings suggest that supplementation of astronauts’ diets with SeM could offer some protection against the expected adverse biological effects resulting from exposure to HZE particle radiation during space travel. Acknowledgments—The authors express sincere appreciation to Dr. John Dicello for sharing his animal holders with us (for use during the HZE particle exposures) and to numerous individuals at the Brookhaven National Laboratory who helped us with these experiments, with special thanks to Dr. William Holley, Dr. Jack Miller, Dr. Betsy M. Sutherland, Dr. Adam Rusek, Dr. R. P. Singh, and Dr. I-Hung Chiang.

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ABBREVIATIONS AGS — Alternating Gradient Synchrotron ATR — an ataxia – telangiectasia-mutated (ATM) and Rad3-related gene BNL — Brookhaven National Laboratory CHK2 — one of the checkpoint effector kinase genes termed Chk2 DCF — 2V,7V-dichlorofluorescein DCFH-DA — 2V,7V-dichlorofluorescein acetate DMEM — Dulbecco’s modified Eagle’s medium EDTA — ethylenediaminetetraacetic acid GAPDH — glyceraldehyde-3-phosphate dehydrogenase HTCA — 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid HZE particle — highly energetic heavy charged particle LET — linear energy transfer PCR — polymerase chain reaction SeM — Selenomethionine