Gastrointestinal Research Group and Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
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Ionizing radiation induces intestinal epithelial
hyporesponsiveness to secretagogues through an unknown mechanism. We
investigated the role of the inducible isoform of nitric oxide (NO)
synthase (iNOS)-derived NO in radiation-induced hyporesponsiveness.
C57BL/6 mice were sham treated or exposed to 10-Gy -radiation and
were studied 3 days later. Tissues were mounted in Ussing-type
diffusion chambers to assess chloride secretion in response to
electrical field stimulation (EFS) and forskolin (10 µM). Transport
studies were also repeated in iNOS-deficient mice. White blood cell
counts were significantly lower in irradiated mice, and there was no inflammatory response as shown by myeloperoxidase activity and histological assessment. iNOS mRNA levels and nitrate/nitrite concentrations were significantly elevated in irradiated colons. iNOS
immunoreactivity localized to the epithelium. Colons from irradiated
wild-type, but not iNOS-deficient, mice exhibited a significant
reduction in the responsiveness of the tissue to EFS and forskolin. The
hyporesponsiveness was reversed by
L-N6-(1-iminoethyl)lysine, 1400W, and
dexamethasone treatments. iNOS-derived NO mediates colonic
hyporesponsiveness 3 days after irradiation in the mouse in the absence
of an inflammatory response.
radiotherapy; secretion; intestine; electrolyte transport; transgenic mice
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INTRODUCTION |
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EXPOSURE TO IONIZING RADIATION is a mainstay of treatment of abdominopelvic malignancies. However, this treatment is associated with a high incidence of untoward gastrointestinal symptoms. The severity of the manifestations of acute gastrointestinal dysfunction may be sufficient to result in interruption or cessation of radiation treatment, which in turn may prevent a curative outcome for the patient (22). Although acute radiation enteritis is usually self-limiting and can be managed symptomatically, severe bouts of acute enteritis are a predictor of more severe, chronic complications months to years after therapy (21, 22, 24).
Nonspecific DNA damage leads to S-phase interruption and cell death in actively dividing cells, which is a well-established consequence of ionizing radiation and mediates its antineoplastic activity (23). However, the high proliferative activity of cells in the crypt region of the intestinal epithelium renders this region a site of bystander injury after abdominopelvic exposure (21). Although the gastrointestinal side effects of exposure to ionizing radiation are generally ascribed to its effect on epithelial mitotic success and subsequent depletion of cells, more recent evidence indicates that epithelial dysfunction is initiated before significant epithelial cell loss. The primary dysfunction observed is a profound hyporesponsiveness to secretagogues in most species studied. Although this phenomenon has been described by us (8, 18) and others (11, 12), the underlying mechanism is not clear. We had, in earlier studies, described a role for mast cells (18), but more recent evidence suggests that mast cells do not mediate the perpetuation of the epithelial dysfunction (8), and effects on the epithelial cell itself have been suggested (16) but by no means unequivocally proven.
Understanding radiation-induced epithelial hyporesponsiveness to secretagogues is critical because the physiologically regulated secretion of chloride and water prevents transepithelial migration of bacteria, bacterial products, or antigens that could trigger an inappropriate and potentially chronic inflammatory response (29). Hyporesponsiveness to secretagogues is characteristic of inflammatory diseases of the gut and has been demonstrated in animal models (19) and human inflammatory bowel disease (14, 25). The exact mechanism underlying this apparently generalized response to inflammation is unclear, but recent data suggest that nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) may be involved. NO is synthesized in numerous cell types, is a known modulator of epithelial function, and has been implicated in models of intestinal inflammation and human disease. In a model of acute colitis in the mouse, there is elevated iNOS expression and activity, as well as epithelial hyporesponsiveness to secretagogues (19). This hyporesponsiveness was partially reversed by pretreatment with a selective iNOS inhibitor. Similar observations have been made in a model of postinflammation dysfunction in a rat model (1). An earlier study showed that after exposure to ionizing radiation, NO is elevated in mouse intestine by an L-arginine-dependent mechanism (27). More recently, we have shown (17) that iNOS levels in the rat intestine were elevated as early as 2 h after radiation treatment.
From the preceding findings we have formulated the hypothesis that radiation-induced epithelial dysfunction, which may predispose the gut to chronic complications, is mediated by iNOS-derived NO. Here we describe the expression, activity, and function of iNOS-derived NO in colons from wild-type and iNOS-deficient mice exposed to therapeutically relevant doses of ionizing radiation. The studies demonstrate a central role for NO in radiation-induced dysfunction.
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METHODS |
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Animals. Male C57BL/6 mice (Charles River; Montreal, QC, Canada), 4-6 wk of age, were housed at a constant temperature (22°C) and photoperiod (12 h light, 12 h dark). Mice were given 5-7 days to acclimatize to these conditions before inclusion in an experiment. They were allowed free access to standard lab chow and tap water. All procedures involving animals were approved by the University of Calgary Animal Care Committee, and experiments were conducted in accordance with the regulations established by the Canadian Council on Animal Care.
Irradiation. Conscious mice were physically restrained in plastic tubes during radiation. Mice were exposed to 10-Gy parallel-opposed field total body radiation from a 60Co therapeutic irradiator at a dose rate of 1.25 Gy/min (Theratron 780-C, Atomic Energy of Canada, Kanata, ON, Canada) at the Tom Baker Cancer Centre (Calgary, AB, Canada). Intestinal mucosal responses to irradiation are dose dependent (11), and this dose corresponds to that which we (18) and others (11) have used previously. Sham-treated mice were placed in the treatment field but were not exposed to the radioactive source. Mice were studied 3 days after irradiation or sham treatment.
White blood cell counts. Aliquots (50 µl) of whole blood were collected from anesthetized mice by cardiac puncture before tissue collection. The blood sample was added to 440 µl of 3% acetic acid and 10 µl of 1% crystal violet. White blood cells were counted in a hemocytometer. A 1-mm2 counting area was used. Cells falling on the center line at the top and right-hand sides were included in the count, whereas the cells on the center line at the bottom and left-hand sides were excluded. Cell counts were expressed as the number of white blood cells/ml of whole blood.
Myeloperoxidase activity. The colon was excised, and a whole thickness sample was taken for determination of myeloperoxidase (MPO) activity as previously described (3). Briefly, 35-100 mg of tissue were homogenized in hexadecyltrimethylammonium bromide phosphate buffer at a concentration of 50 mg/ml. The samples were then centrifuged, and the supernatant was assayed. The reaction between o-dianisidine and the oxygen produced by the peroxidation of H2O2 produced a colorimetric product in a 1-to-1 ratio that was measured at 450 nm on a UVmax kinetic microplate reader (Molecular Devices; Sunnyvale, CA) using SOFTmax version 2.34 computer software (Molecular Devices).
RT-PCR.
RT-PCR was conducted as we described previously (19). Briefly, the
colon was excised and a 50- to 100-mg segment was placed in 1 ml of
TRIzol reagent (Life Technologies; Grand Island, NY) for RNA
extraction. Tissue samples were stored in TRIzol at 70°C until processed for RNA extraction. Tissue was thawed and homogenized in TRIzol, and 0.2 ml of chloroform was added to the tube. Samples were
centrifuged at 10,500 rpm for 30 min at 4°C. An aliquot (500 µl)
of the aqueous phase was removed and mixed with 500 µl of isopropanol
to precipitate RNA. The samples were centrifuged at 12,000 g
for 20 min. The RNA pellet was then washed in 75% ethanol and
centrifuged for 5 min at 7,500 g. The RNA pellet was
redissolved in 100 µl of ultrapure autoclaved water. The purity and
concentration of the RNA were measured using a Gene Quant II nucleic
acid analyzer (Pharmacia Biotech, Uppsala, Sweden). RNA (2 µg) was
added to a reaction mixture containing 2 µl of 10× PCR buffer,
2 µl of 10 mM dNTPs, 2 µl N6, and 0.5 µl of RNAguard.
Superscript enzyme (300 units) was added for reverse transcription
(RT). RNA samples were first incubated for 10 min, and the reaction
mixture was heated to 42°C for 50 min and then to 95°C (to
destroy the Superscript enzyme) in a Temptronic thermal cycler
(Thermolyne, Kirkland, WA). PCR was carried out on the cDNA from the RT
reaction at 28 cycles for iNOS using rat 5' and 3' iNOS
primers and 22 cycles for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), an internal control. Sequences of primers are those we
previously published (19). The PCR samples were separated on a 1%
agarose gel containing ethidium bromide for 5 min at 100 V and 40 min
at 80 V. The gel was photographed using a Bio-Rad Gel Doc 2000 (Bio-Rad, Hercules, CA), and densitometry was performed using Quantity
One version 4.0.1 software (Bio-Rad).
HPLC. Nitrite and nitrate levels were determined by HPLC as previously described (20). Briefly, the colon was excised and washed and incubated in 1 ml of PBS. The samples were incubated for 10 min at 37°C and then homogenized and centrifuged at 12,000 g for 10 min. The supernatant was taken for HPLC. The supernatant was centrifuged at 14,000 g for 2 min to remove any particulate matter. The samples were then diluted in a 1-to-3 ratio with HPLC-grade water. The samples were deproteinated by ultrafiltration through nitrocellulose filters (Centrisart C-4 ultrafiltration tubes, mol wt cutoff: 5,000; Sartorius, Germany) and then centrifuged (6,000 g, 60 min). An aliquot of the filtrate (100 µl) was taken and analyzed for nitrite and nitrate content. Nitrite and nitrate eluted as separate peaks. Concentrations of nitrite and nitrate were added together to give an estimate of NO concentration.
Electrolyte transport experiments. Mice were killed by cervical dislocation. The colonic tissue was rapidly excised and placed in ice-cold (4°C) Krebs buffer (in mM: 115 NaCl, 2.0 KH2PO4, 2.4 MgCl2, 1.3 CaCl2, 25.0 NaHCO3 and 8.0 KCl) bubbled with carbogen (5% CO2-95% O2). Feces were removed, and the colon was cut along the mesenteric border. The tissue was mounted in Ussing-type diffusion chambers (Navicyte, Sparks, NV) and bathed on both the serosal and mucosal sides with Krebs buffer (pH 7.4, 37°C) containing 10 mM glucose (pH 7.4). Buffers were circulated and oxygenated with a gas lift system using 95% O2-5% CO2. A voltage-clamp apparatus (EVC-4000, World Precision Instruments, Sarasota, FL) was used to maintain the potential difference across the tissue at 0 V. The short-circuit current (Isc) required to clamp the tissue was used as a measure of active electrogenic ion transport across the epithelium. Isc was recorded using a digital data acquisition system (MP100, Biopac Systems, Santa Barbara, CA) and analysis software (AqcKnowledge, version 3.2.6, Biopac). The tissue conductance (Isc/potential difference) was measured as an indicator of epithelial ionic permeability. Electrical field stimulation (EFS), which depolarizes secretomotor neurons in the preparation to cause chloride secretion (5), was delivered via platinum wires inserted into each bathing solution. The stimulation parameters were stimulus duration 3 s, stimulus strength 50 V, frequency 10 Hz, and pulse duration 500 µs. Once baseline Isc had been reestablished, tissues were exposed serosally to the cAMP-dependent secretagogue forskolin (10 µM) and the change in Isc was measured. To assess the role of iNOS in tissue hyporesponsiveness, experiments were conducted in which the selective iNOS inhibitors L-N6-(1-iminoethyl)lysine (L-NIL, 3 µM; Ref. 6) or 1400W (5 µM; Ref. 9) were applied serosally. After a 10-min incubation with either L-NIL or 1400W, responses to EFS and forskolin were measured.
Histology. For immunohistochemistry, segments of mouse colon were removed and fixed in Zamboni's fixative overnight. They were then washed and stored in PBS with azide and cryoprotected in PBS containing sucrose. Sections (12-14 µm) were cut using a cryostat (Microm) and placed onto poly-D-lysine-coated microscope slides. Sections were washed three times in PBS and were incubated with a rabbit anti-iNOS primary antibody (Transduction Laboratories; Lexington, KY) at 4°C overnight. Sections were then rinsed with PBS and incubated with donkey anti-rabbit CY3 conjugated secondary antibody (Jackson Laboratory, Bar Harbor, ME) for 90 min. They were then washed three times in PBS, coverslipped, and viewed under epifluorescence using a Zeiss Axioplan microscope. Images were captured and digitized with a charge-coupled device video camera and Northern Exposure imaging software (Carson Vision, Edmonton, AB, Canada).
Drugs. Routine buffer reagents were obtained from BDH (Toronto, ON, Canada) unless indicated otherwise. 1400W was obtained from Alexis Biochemicals (San Diego, CA). Dexamethasone sodium phosphate was obtained from Sabex (Boucherville, QC, Canada). Other chemicals were obtained from Sigma Chemical (Mississauga, ON, Canada).
Statistics. Data are expressed as means ± SE. Statistical analyses were conducted using GraphPad Instat software (version 3.0, GraphPad Software, San Diego, CA). One-way ANOVA with a post hoc Tukey test was used to compare more than two groups. Unpaired Student's t-test was used to compare two groups. A probability (P) value of <0.05 was considered significant.
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RESULTS |
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Response to irradiation.
Sections of colon were assessed histologically to determine whether
there were any morphological changes in the colon 3 days after exposure
to 10-Gy -radiation. At the light microscopic level, there were no
differences between the sections taken from the sham-treated animals
and those from the irradiated animals (data not shown). Crypt
architecture was preserved in all cases, and the epithelium was
structurally intact, other than occasional sectioning artifacts
observed in both sham-treated and irradiated tissue. There was no
obvious inflammatory cell infiltrate into the mucosae of colons from
irradiated mice.
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Expression of iNOS.
Expression of iNOS mRNA, as determined by RT-PCR, was induced in
colonic segments taken from irradiated mice (Fig.
1). Expression of iNOS in control animals
was either absent or was present at very low levels in colonic tissue
from sham-treated mice (n = 9). Densitometry showed a
significant upregulation of iNOS expression in irradiated colon
(n = 7) compared with controls (Fig.
2). Not surprisingly, iNOS expression was
not observed in colonic segments from either sham-treated or irradiated
iNOS-deficient (iNOS/
)
mice (n = 6 per group;
C57BL/6-Nos2tm1Lau, backcross generation
N10; Jackson Labs, Bar Harbor, ME; Fig. 1). Optical density of the PCR
product of GAPDH, which was run as the "housekeeping" gene, was
not significantly different between sham and irradiated groups
(n > 6 per group).
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Electrolyte transport studies.
Colonic segments from sham-treated and irradiated mice were mounted in
diffusion chambers and voltage clamped after challenge with EFS and
forskolin. Exposure to EFS and forskolin both caused an increase in
Isc, which was consistent with an increase in the serosal-to-mucosal flux of chloride (5). The responses were transient,
peaking within 5 min and returning to the original baseline within 10 min. However, the maximal Isc responses to both EFS
and forskolin were significantly lower in colonic tissues from
irradiated mice (sham, n = 8, IR, n = 6)
(Fig. 4). Acute inhibition of iNOS activity
with either of the selective iNOS inhibitors, L-NIL or
1400W, reversed the hyporesponsiveness of the irradiated tissue to EFS
and forskolin (sham + vehicle, n = 6; sham + L-NIL, n = 8; sham + 1400W, n = 6; IR + vehicle, n = 9; IR + L-NIL, n = 7, IR + 1400W, n = 6) (Fig. 4). Similarly, pretreatment of mice with
dexamethasone, which blocks iNOS expression (7), reversed the
radiation-induced hyporesponsiveness to EFS and forskolin (sham + saline, n = 6; sham + Dex, n = 6; IR + saline, n = 4; IR + Dex, n = 6) (Fig.
5).
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DISCUSSION |
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In this study we have shown that, 3 days after exposure to a single
10-Gy dose of -radiation, the mouse colon is hyporesponsive to EFS
and forskolin compared with sham-treated controls in vitro. This
hyporesponsiveness is caused, as in models of colitis, by iNOS-derived
NO. Of particular interest in the present study is the fact that
NO-induced epithelial hyporesponsiveness occurs in the absence of an
inflammatory response.
We initially hypothesized that the hyporesponsiveness observed after irradiation was caused by the induction of an inflammatory response. Intestinal inflammation, such as that triggered in animal models of inflammatory bowel disease, or in human inflammatory bowel disease, is associated with epithelial hyporesponsiveness to secretagogues (10, 13, 19). Furthermore, local abdominopelvic irradiation of anesthetized rats was accompanied by a rapid neutrophil influx into the intestinal submucosa and mucosa (4). One of the crucial findings of the present study, however, is the fact that the hyporesponsiveness observed after irradiation is not associated with an inflammatory response. Several pieces of evidence support this. First, there was no inflammatory cell influx observed in histological sections of colon from irradiated mice, at least at 3 days after 10-Gy radiation (data not shown). Our observations at 3 days after irradiation do not preclude granulocyte influx at an earlier time point after irradiation, although it is unlikely that this would occur to any appreciable degree and still be completely resolved in 3 days. Histological assessment of colonic samples after 10-Gy radiation confirmed that, at least at the light microscopic level, the epithelium was structurally intact and the normal crypt architecture was observed. Thus significant cell loss and epithelial denudation does not account for the observed hyporesponsiveness at this time point. Similarly, epithelial patency was observed after irradiation in the rat small intestine (18).
Second, colonic MPO activity, which is a measure of granulocyte infiltration into the tissue, was not different between sham-treated and irradiated mice (Table 1). Our previous studies (18) have shown that in the rat ileum and colon hyporesponsiveness to EFS after irradiation occurs in the absence of an acute inflammatory response as measured by MPO. The difference between our studies and that of Buell and Harding (4), which showed a rapid inflammatory influx into the tissue, is the mode of irradiation used. We have treated animals with whole body irradiation, whereas their studies were in animals receiving local abdominopelvic exposure, which would leave sufficient hematopoietic tissue unaffected to allow for granulocyte repopulation and subsequent recruitment into the injured site.
Finally, in our studies, whole body exposure of mice to 10-Gy
-radiation was associated with a white blood cell count that was
significantly depleted at 3 days, suggesting that there was a reduced
pool of potential inflammatory cells available for recruitment to sites
of injury. Taken together, these results suggest that a
radiation-induced inflammatory response was not a contributing factor
in the hyporesponsiveness of the tissue to secretagogues in vitro at
this time point.
Of note in this study is the observation that radiation-induced epithelial hyporesponsiveness is mediated by iNOS-derived NO, despite the lack of an inflammatory response. Increased expression of iNOS mRNA was associated with increased production of NO, as shown by HPLC measurement of nitrate and nitrite levels in the tissue (Fig. 1). Inhibition of iNOS activity with the iNOS inhibitors L-NIL and 1400W, at doses that have been shown to be selective for iNOS (9, 19), returns the Isc response to EFS and forskolin to levels similar to those observed in the sham-treated mice (Fig. 4). The results further demonstrate an ongoing release of iNOS-derived NO, because the inhibitors were added to the bath only 10 min before challenge with secretagogue. A similar reversal of hyporesponsiveness was observed in mice pretreated with dexamethasone, which prevents induction of iNOS (7). The conclusion that iNOS is involved in the epithelial hyporesponsiveness is further supported by the studies in iNOS-deficient mice, which did not exhibit radiation-induced increase in conductance or hyporesponsiveness to EFS or forskolin. NO has been shown to inhibit secretory responses in rat small intestine in vivo (2, 26). Our previous studies revealed an iNOS dependency of inflammation-induced hyporesponsiveness, both during acute inflammation in mouse colon (19) and 6 wk after a bout of colitis in the rat (1). The present observation that iNOS-derived NO mediated epithelial hyporesponsiveness to secretagogues in the absence of an inflammatory response is important in that it suggests that generation of NO represents a common mechanism underlying the inability of the epithelium to respond to secretagogues in a number of clinical situations.
Our data have indicated an apparent difference in responsiveness of
sham-treated wild-type and iNOS-deficient mice. Although responses to
EFS were reduced in the colons from
iNOS/
mice, responses to
forskolin were not. If the
iNOS
/
mice were basally
hyporesponsive in the same sense or through the same mechanism that
irradiated wild-type mice were hyporesponsive, then we would have
expected reduced Isc responses to both EFS and
forskolin. Given the difference in responsiveness to EFS and forskolin
in the iNOS
/
mice, we suggest
that perhaps the iNOS
/
mice
exhibit a different degree of enteric neural control of epithelial
function. Differences, as yet undescribed, may exist between these
strains with respect to enteric nervous system "wiring" and/or
function. If this is the case, then our data exemplify the caution that
must be exercised in comparing physiological data derived from
wild-type versus genetically deficient animals.
The cellular source of iNOS that contributes to radiation-induced
hyporesponsiveness to secretagogues is not clear, but the data
presented here (Fig. 3) suggest that it is localized to the epithelium.
In some mice, epithelial expression was restricted to the surface
epithelial cells, whereas in other mice, expression was observed in
both surface and crypt cells. We have previously shown (17) that whole
body exposure of rats to 10-Gy -radiation resulted in induction of
iNOS mRNA in ileum and colon as early as 2 h after irradiation. In the
rat, however, expression returned to control levels by 24-48 h,
whereas in the present study we still have evidence of iNOS induction
at 3 days after exposure. We have also found that, contrary to the rat,
in which radiation-induced iNOS immunoreactivity is always localized to
epithelial cells along the length of the crypt-villus axis, iNOS in
irradiated mouse colon is found primarily in the apical epithelial
cells. The secretion of chloride and water by the colonic epithelium is
thought to be localized to the relatively undifferentiated crypt
enterocytes (28). Clearly, in those mice in which iNOS expression is
present in the crypt, NO may act on chloride-secreting enterocytes in a
paracrine, or even autocrine, manner. Furthermore, it is possible that
NO could still affect crypt function in those mice in which iNOS
immunoreactivity was confined to the surface epithelial cells. NO,
although a free radical, is relatively unreactive, and this, along with
its hydrophobicity, may allow it to have effects several cells away
from its site of production. Furthermore, the mucosal microcirculation
could provide a means of transport for apically derived NO to the crypt region.
The implications for epithelial hyporesponsiveness are important when one considers the host defense role played by secretion. The secretion of chloride and, hence, water into the crypt lumen prevents or limits the translocation of bacteria, bacterial products, or antigens into the lamina propria, where they may trigger or perpetuate an inflammatory response (29). Change in epithelial barrier function, which also includes increased permeability, is a predictor of and likely contributes to relapses of inflammation in Crohn's disease (30). Given the link between the acute and chronic intestinal manifestations of radiation exposure (21) and given the participation of epithelial dysfunction in development of inflammatory conditions in the gut, there is a clear impetus to discover the mechanisms of, and therapeutic interventions for, radiation-induced epithelial hyporesponsiveness to secretagogues.
The mechanism whereby NO reduces epithelial responsiveness to
secretagogues after irradiation remains to be determined. Recently, it
was shown that NO reduced the expression of the -subunit of Na+-K+-ATPase in the MTAL kidney cell line
(15). Furthermore, Na+-K+-ATPase activity is
reduced in colonic epithelial cells after irradiation (16).
Na+-K+-ATPase provides the electrochemical
gradient that favors chloride flux into the enterocyte, with subsequent
secretion through the apical chloride channel. A link among radiation,
NO, and Na+-K+-ATPase has not been described.
Further studies will be required to more fully explore the effect of NO
on radiation-induced epithelial secretory dysfunction.
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ACKNOWLEDGEMENTS |
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The authors thank Jane Wiggins (Tom Baker Cancer Centre, Alberta Cancer Board, Calgary, AB) for calculation of radiation dose and operation of the Theratron irradiator, Dr. Marcello Muscará for measuring nitrogen oxides by HPLC, and Dr. Keith Sharkey for assistance with iNOS immunohistochemistry.
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FOOTNOTES |
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This work was supported by a grant (MT-14660) from the Medical Research Council of Canada. W. K. MacNaughton is an Alberta Heritage Foundation for Medical Research Scholar.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. MacNaughton, Dept. of Physiology and Biophysics, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 4N1 (E-mail: wmacnaug{at}ucalgary.ca).
Received 17 August 1999; accepted in final form 28 October 1999.
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