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The effect of heme oxygenase-1 induction by glutamine on radiation-induced intestinal damage: the effect of heme oxygenase-1 on radiation enteritis
The American Journal of Surgery 191 (2006) 503–509
Scientific papers
The effect of heme oxygenase-1 induction by glutamine on radiation-induced intestinal damage: the effect of heme oxygenase-1 on radiation enteritis Murat Giris¸, M.S.C.a, Yes¸im Erbil, M.D.b,*, Serdar Öztezcan, M.D.a, Vakur Olgaç, Ph.D.c, Umut Barbaros, M.D.b, Ug˘ur Deveci, M.D.b, Banu Kirgiz, M.D.a, Müjdat Uysala, Gülçin Aykaç Tokera a
b
Department of Biochemistry, Istanbul University, Istanbul Medical Faculty, Capa, Istanbul, Turkey Department of General Surgery, Istanbul University, Istanbul Medical Faculty, 34340 Capa, Istanbul, Turkey c Department of Pathology, Istanbul University, Istanbul Medical Faculty, Capa, Istanbul, Turkey Manuscript received May 20, 2005; revised manuscript November 3, 2005
Abstract Background: Radiation enteritis is a significant clinical problem in patients receiving ionizing radiation directed at the abdomen or pelvis. The small intestine is the most radiosensitive gastrointestinal organ. Myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels of the small intestine were measured to determine the oxidative damage caused by radiation. In addition, caspase-3 activity of the small intestine was measured to define the degree of apoptosis. The present study was undertaken to investigate the effect of glutamine administration on heme oxygenase-1 (HO-1) expression of the radiation enteritis model. Methods: Rats received 1 g/kg/d glutamine (HO-1–inducer) for 7 days before irradiation and continued for 3 days after irradiation. Zn-prothoporphyin (Zn-PP) 40 mol/kg was delivered subcutaneously for 1 day before irradiation. Intestinal MPO activities and MDA levels are indicators of oxidative damage, whereas caspase-3 activities show the degree of apoptosis of the small intestine. At histopathologic examination, terminal ileum tissue was analyzed for morphologic changes. Also, the nuclear factor- (NF-) expression level of the terminal ileum was determined with immunohistochemisty methods to show the mucosal inflammatory process. Results: Irradiation significantly increased the intestinal MPO and caspase-3 activities, MDA levels, and HO-1 expression in comparison with the sham group. Glutamine treatment was associated with increased HO-1 expression, decreased MPO activity, caspase-3 activity, and MDA levels. Inhibition of HO-1 activity by Zn-PP completely eliminated the protective effects of glutamine. Histopathologic examination showed that the intestinal mucosal structure was preserved in the glutamine-treated group. In the irradiation group, NF-B overexpression was detected. NF-B positivity was strongest in the intestine of animals in the radiation alone group and the Zn-PP–treated irradiation group. Conclusions: Glutamine appears to have protective effects against radiation-induced intestinal damage. This protective effect is mediated in part by the induction of HO-1 activity because inhibition of Zn-PP resulted in the complete abolishment of the protective effect of glutamine. © 2006 Excerpta Medica Inc. All rights reserved. Keywords: Heme oxygenase-1; Glutamine; Zn-prothoporphyin; Irradiation; Apoptosis; NF-B
Radiation enteritis occurs during radiotherapy for many intraabdominal and pelvic cancers such as cervical, endometrial, ovarian, bladder, prostate, and rectal [1,2]. Abdominal irradiation causes mucosal damage in the gastrointestinal epithelium with activated inflammatory cells. The mucosal damage was described as destruction of crypt cells, a decrease in villous
* Corresponding author. Tel.: ⫹90-212-5331784; fax: ⫹90-212-6319771. E-mail address: [email protected]
height, and a decrease in the number, ulceration, and necrosis of the gastrointestinal epithelium [2– 4]. Although the pathogenesis of radiation enteritis is not clear, it is presumed to be an inflammatory process in which various mediators such as eicosanoids, cytokines, and reactive oxygen metabolites take place [4 –7]. Heme oxygenase (HO) is an enzyme that catalyzes the first and rate-limiting step in heme degradation, which yields the byproducts carbon monoxide (CO), free iron, and biliverdin, all of which possess free radical scavenging
0002-9610/06/$ – see front matter © 2006 Excerpta Medica Inc. All rights reserved. doi:10.1016/j.amjsurg.2005.11.004
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properties. HO-1 induction has been considered to protect cells against oxidative injuries [8 –12]. Glutamine is the respiratory fuel for the mucosal cell of the intestine. During pathologic conditions, the consumption of glutamine increases. In addition, glutamine has been shown to be beneficial in the prevention of mucosal integrity [13–16]. The mechanism of protection by glutamine remains unclear. In the present study we investigated whether HO-1 induction by glutamine could protect radiation-induced damage in the intestinal tissues by examining intestinal myeloperoxidase (MPO) activities and malondialdehyde (MDA) levels as indicators of oxidative damage and caspase-3 activitiy as an apoptosis marker. At histopathologic examination, the terminal ileum tissue was analyzed for morphologic changes. Also, the nuclear factor (NF-) expression level of the terminal ileum was determined with immunohistochemical methods to show the mucosal inflammatory process.
Materials and Methods Animals Fifty-six male Wistar-albino rats weighing 250 to 300 g (Istanbul University, Institute of Experimental Medicine and Research, Istanbul, Turkey) were used in the study. All animals were housed in wire-mesh– bottomed cages in a 12 hour light-dark cycle. Rats were kept in a room at a constant temperature of 22°C ⫾ 2°C and were fed a standard chow diet and water. The study was approved by the Ethics Committee of Istanbul University, Istanbul Medical School. Study design The rats were divided into 8 groups. Group 1 (abdominal irradiation [RT]; n ⫽ 8): abdominal irradiation alone; group 2 (RT/glutamine [Glu]; n ⫽ 8): abdominal irradiation and glutamine (Sigma, St. Louis, MO) 1 g/kg/d intragastric gavage for 7 days before irradiation and 3 days after irradiation; group 3 (RT/Zn-PP; n ⫽ 8): abdominal irradiation and zinc-prothoporphyin (Zn-PP; Sigma) 40 mmol/kg subcutaneously 1 day before radiation; group 4 (RT/Glu/Zn-PP; n ⫽ 8): abdominal irradiation and glutamine and Zn-PP; group 5 (Glu; n ⫽ 6): glutamine alone 1 g/kg/d intragastric gavage for 10 days; group 6 (Zn-PP; n ⫽ 6): Zn-PP alone 40 mmol/kg subcutaneously once; group 7 (Glu/Zn-PP; n ⫽ 6): Zn-PP and glutamine alone; group 8 (sham control group; n ⫽ 6): isotonic saline solution 1 cc/rat intragastric for 10 days. Glutamine was administered 7 days before radiation and 3 days after irradiation. All animals were irradiated on day 7 and killed on day 10. Zn-PP was dissolved in .1 mL of .5 mol/L NaOH; normal saline was added until the drug preparation equaled .5 mL and the pH was adjusted to 7.4,
and was stored in the dark to prevent photodegradation. Zn-PP was administered 1 day before radiation. Irradiation consisted of a single dose of 1,000 cGy (source to skin distance, 80 cm) to the abdominal region of the animals anesthetized with sodium thiopental 40 mg/kg intraperitoneally. Three days after irradiation, all animals were killed with a lethal dose of sodium thiopental. The small intestine then was removed and washed with ice-cold isotonic saline solution. Samples of the terminal ileum were fixed in buffered formalin for histopathologic examination. The rest of the tissue was stored at ⫺80°C until it was processed for biochemical analysis. MPO activity Intestinal MPO activity was assayed by the o-dianisidine method [17]. Tissue samples were suspended in 50 mmol/L, pH 6.0, potassium phosphate buffer containing .5% hexadecyltrimethylammonium bromide and homogenized. A sample of homogenate was sonicated for 20 seconds, freezethawed 3 times, sonicated again for 10 seconds, and centrifuged at 15,000 ⫻ g for 10 minutes at 4°C. The reaction was started by mixing and incubating the 100 L of the supernatant at 25°C with a solution composed of 2.810 L of 50 mmol/L potassium phosphate buffer, pH 6.0, 30 L of 20 mg/mL o-dianisidine dihydrochloride, and 30 L of 20 mmol/L H2O2. After 10 minutes the reaction was terminated by the addition of 30 L of 2% sodium azide. The change in absorbance was read at 460 nm and the results were calculated by using the molar absorptivity coefficient of oxidized o-dianisidine (1.0062 ⫻ 104 mol/L⫺1 cm⫺1). MPO activity (1 unit) was expressed as the amount of enzyme necessary for the degradation of 1 mol of H2O2/min/100 mg tissue at 25°C. MDA assays The levels of MDA in the tissues were measured to assess lipid peroxidation. Samples of the small intestine were homogenized with ice-cold 150 nmol/L potassium chloride for the determination of MDA levels. MDA levels were measured spectrophotometrically. Results are expressed as nmol of MDA per gram of tissue [18]. Caspase activity Caspase-3 activity was measured by quantifying the cleavage of acetyl-Asp-Glu-Val-Asp p-nitroaniline with a colorimetric caspase-3 assay kit (Sigma). P-nitroaniline released by the enzymatic hydrolysis of acetyl-Asp-Glu-ValAsp p-nitroaniline was calculated from a p-nitroaniline standard carve and the molar absorptivity coefficient of p-NA at 405 nm (10,500 mol/L⫺1 cm⫺1). The supernatant obtained by the centrifugation of 10% tissue homogenate at 15,000 ⫻ g for 5 minutes at 4°C was used as an enzyme source and
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incubated with and without caspase-3 inhibitor, Ac-DEVCHO, according to the assay protocol of the kit. Caspase activity was calculated by substracting the absorbance measured in the presence of substrate plus inhibitor from the absorbance observed by incubating with substrate alone for 20 minutes at 37°C. The activity of the enzyme was expressed as p-nitroaniline liberated per mg protein per minute. The protein concentration of the enzyme source was measured by using bicinchoninic acid [19]. Western blot analysis for HO-1 Western blot analysis was performed with modifications as previously described [8]. Briefly, livers were homogenized in the 5 volume lysis buffer containing 10 mmol/L Tris-HCl (pH 8), 140 mmol/L NaCl, .5 mmol/L ethylenediaminetetraacetic acid, 1% TritonX-100, 1% deoxycholate, .1% sodium dodecyl sulfate, .5 mmol/L phenylmethylsulfonyl fluoride, 1 g/L leupeptin, and 1 g/L aprotinin. The homogenate was centrifuged at 10,000 ⫻ g for 15 minutes at 4°C. The supernatant was centrifuged at 105,000 ⫻ g for 1 hour at 4°C. The precipitated microsome was dissolved in the earlier-described Tris-HCl solution. Equal amounts of protein (100 g) were loaded onto 12% sodium dodecyl sulfate–polyacrylamide gels and blotted onto the polyvinylidine difluoride membrane (Millipore, MA). Nonspecific binding sites were blocked with Trisbuffered saline–Tween 20 containing 5% nonfat dry milk overnight at 4°C. Membranes were incubated in polyclonal antibody against rat HO-1 at 1:1,500 dilutions and horseradish-peroxidase– conjugated secondary antibody (goat anti-rabbit IgG; Stress Gen, Toronto, Canada) at 1:16,000 dilutions. The membrane-bound antibody was visualized with the enhanced chemiluminescence Western blotting detection system kit (Amersham, Pittsburgh, PA). Bands were quantitated using a densitometer (image analysis software, Vilber Lourmat Biotechnology, Marne-la-Vallée, France).
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hematoxylin. The staining intensity was defined in percentages and was given scores ranging from 1 to 4 (⬎75%, 4⫹; 50%–75%, 3⫹; 25%–50%, 2⫹; ⬍25%, 1⫹. Statistical analysis All data were expressed as mean ⫾ SD. The MannWhitney U test and analysis of variance were used for statistical analysis and a P value of less than .05 was considered significant (SPSS 11.0 for Windows, Chicago, IL). Results All rats developed radiation-induced diarrhea within 24 hours after abdominal radiation. There were no treatmentrelated mortalities. HO-1 protein expression Fig. 1 shows that treatment with glutamine in the irradiated rats significantly induced HO-1 expression when compared to sham control rats (49 ⫾ 8 vs. 15 ⫾ 4). Irradiation similarly induced HO-1 expression (30 ⫾ 5). HO-1 expression seen in the group given Zn-PP was higher than in the control group. This was believed to be the result of an increase in HO-1 expression on a molecular basis to compensate for the decrease in HO-1 activity. The increase in MPO and MDA levels also is proof of a Zn-PP–suppressor effect on HO-1 activity. MPO activity In all irradiated rats, intestinal MPO activity was found to be increased significantly compared with the sham con-
Histopathology The terminal ileum tissue was analyzed for morphologic changes. The changes in the terminal ileum mucosa were evaluated for mean villous height and the number of villous per unit by micrometer-adapted light microscope. NF-B expression All specimens were fixed in 10% buffered formalin. Paraffin blocks prepared from routinely processed specimens were cut into 5 slices and were deparaffinized. Antigen retrieval was performed after this process. After microwave incubation of the peroxyblock, followed by ultra V block procedure, primary antibodies (NF-B neomarkers anti-rabbit P50AB-2) were applied. After this process, biotinlated secondary antibody, streptovidin peroxidase, and substrat-cromogen (amino ethyl carbasole) solution was applied, respectively. Nuclear staining was performed with
Fig. 1. Intestinal HO-1 protein expression in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .05; a and c is P ⬍ .05; a and d is P ⬍ .001; b and d is P ⬍ .001; c and d is P ⬍ .001.
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Fig. 2. Intestinal MPO activity in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .001; a and c is P ⬍ .001; b and c is P ⬍ .001.
trol group rats (P ⬍ .001, 6.9 ⫾ 1.2 Ug/tissue vs. 2.1 ⫾ .4 Ug/tissue). Intestinal MPO activity was significantly lower in the glutamine-treated irradiation group compared with the group receiving only irradiation (P ⬍ .001, 2.3 ⫾ .2 Ug/tissue vs. 6.9 ⫾ 1.2 Ug/tissue). In the Zn-PP–treated irradiation group, intestinal MPO activity (11.9 ⫾ 2.2 Ug/ tissue) was found to be increased significantly compared with all other groups including the group receiving only irradiation (P ⬍ .001). However, Zn-PP alone did not change MPO activity compared with the other control groups (Fig. 2). MDA levels The MDA level of the intestinal tissues was found to be significantly higher in the irradiated rats compared with the sham group rats (1.9 ⫾ .2 nmol MDA/mg protein vs. .9 ⫾ .1 nmol MDA/mg protein, P ⬍ .001). Treatment with glutamine significantly decreased the MDA levels compared with the group receiving only irradiation (.8 ⫾ .2 nmol MDA/mg protein vs. 1.9 ⫾ .2 nmol MDA/mg protein, P ⬍ .001). In the Zn-PP–treated irradiation group, intestinal MDA activity was found to be decreased significantly compared with the group receiving only irradiation (1.7 ⫾ .1 nmol MDA/mg protein vs. 1.9 ⫾ .2 nmol MDA/mg protein), but increased significantly compared with the other groups (P ⬍ .001). However, Zn-PP alone did not change MDA activity compared with the other control groups (Fig. 3).
Fig. 3. Intestinal MDA levels in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .001; a and c is P ⬍ .001; b and c is P ⬍ .001.
only irradiation (4.3 ⫾ 1.2 nmol mg protein/min vs. 13.2 ⫾ 1.9 nmol mg protein/min, P ⬍ .05). In the Zn-PP–treated irradiation group, intestinal caspase-3 activity (16.9 ⫾ 3.2 nmol mg protein/min) was found to be increased significantly compared with all other groups including the group receiving only irradiation (P ⬍ .001). However, Zn-PP alone did not change caspase-3 activity compared with the other control groups (Fig. 4). Histopathologic assessment Ileal villous number and height were found to be significantly lower in irradiated rats compared with sham group rats (4.6 ⫾ .6 and .14 ⫾ .01 mm vs. 9.1 ⫾ .9 and .48 ⫾ .05 mm, respectively, P ⬍ .001). Glutamine pretreatment before irradiation significantly prevented a decrease in villous number and height (7.9 ⫾ .5 and .33 ⫾ .01 mm,
Caspase-3 activity The caspase-3 activity of the intestinal tissues was found to be significantly higher in the irradiated rats compared with the sham group rats (13.2 ⫾ 1.9 nmol mg protein/min vs. 3.4 ⫾ .2 nmol mg protein/min, P ⬍ .001). Glutamine administration significantly decreased the caspase-3 activity of the intestinal tissues compared with the group receiving
Fig. 4. Intestinal caspase-3 activitiy in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .001; a and c is P ⬍ .05; a and d is P ⬍ .001; b and d is P ⬍ .05; c and d is P ⬍ .001.
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Fig. 7. Mean ⫾ SD ratio of NF-B expression of the rat intestines in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .001; a and c is P ⬍ .05; a and d is P ⬍ .001; b and c is P ⬍ .05; b and d is P ⬍ .05.
respectively). In the Zn-PP–treated irradiation group, villous number and height were found to be decreased significantly (3.6 ⫾ .8 and .13 ⫾ .03 mm, respectively) compared with all groups other than the group receiving only irradiation (P ⬍ .001) (Figs. 5 and 6).
Fig. 5. Mean ⫾ SD of (A) number of villi and (B) villous height in all groups. Letters signify different values according to analysis of variance testing. (A) The significance between a and b is P ⬍ .001; a and c is P ⬍ .05; a and d is P ⬍ .05; b and c is P ⬍ .05; b and d is P ⬍ .05. (B) The significance between a and b is P ⬍ .05; a and c is P ⬍ .05; b and c is P ⬍ .05.
NF-B expression NF-B positivity was strongest in the intestines of animals in the group receiving only irradiation and the Zn-PP– treated irradiation group. NF-B expression was decreased in the glutamine-treated group (Figs. 7 and 8). Comments The results of the present study suggest that oxidative damage, apoptosis, and HO-1 activity contribute to the
Fig. 6. (A) Distal ileum from irradiation group. There is villous shortening and the villous epithelium has been damaged completely, hematoxylin-eosin ⫻ 40. (B) Distal ileum from glutamine-treated irradiation group. There is pronounced epithelial damage that has been preserved partly, hematoxylin-eosin ⫻ 40. (C) Distal ileum from the Zn-PP–treated irradiation group. The mucosal epithelium has been damaged completely, hematoxylin-eosin ⫻ 40. (D) Distal ileum from the glutamine- and the Zn-PP–treated irradiation group. The mucosal epithelium has been preserved partly, hematoxylin-eosin ⫻ 40. (E) Distal ileum from glutaminealone group. (F) Distal ileum from Zn-PP alone group. (G) Distal ileum from glutamine and Zn-PP group. (H) Distal ileum from control group. Last four groups are seen normal intestinal histologic findings, hematoxyline-eosin ⫻ 100.
Fig. 8. (A) Nuclear kappa factor-B (NF-B) overexpression in the intestine in the irradiation group, NFB ⫻ 40. (B) Decreased NF-B expression in the intestine in the glutamine-treated irradiation group, NFB ⫻ 40. (C) NF-B overexpression in the intestine in the Zn-PP–treated irradiation group, NFB ⫻ 40. (D) Decreased NF-B expression in the intestine in the glutamine- and Zn-PP–treated irradiation group, NFB ⫻ 100. (E) Distal ileum from glutamine-alone group. (F) Distal ileum from Zn-PP alone group. (G) Distal ileum from glutamine and Zn-PP group. (H) Distal ileum from control group. In the last four groups, NF-B expression was not seen. Normal intestinal histologic findings are seen, NF-B ⫻ 100.
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development of radiation enteritis after abdominal radiation, as assessed by increased lipid peroxidation, MPO activity, caspase-3 levels, and HO-1 induction in the intestinal tissue. Our results also show that glutamine protects against radiation-induced intestinal damage. The protective effect of glutamine is related in part to site-specific HO-1–inducing activity in the intestinal mucosa, which leads to the reduction of tissue inflammation and the suppression of apoptosis. Radiation enteritis occurs as a response to abdominal radiation, which can cause mucosal damage in the gastrointestinal mucosal epithelium. The small intestine is one of the most radiosensitive organs in the abdomen, and in the present study we have shown a significant decrease in the number and height of the intestinal villi of the small intestine by histopathologic examination in irradiated animals [5–7]. This finding is consistent with radiation-induced damage and inflammation. Radiation also can activate inflammatory cells, leading to the synthesis and release of certain cytokines, inflammatory mediators, and reactive oxygen metabolites [20 –22]. In our study, in all irradiated rats, the intestinal MPO activity was found to be increased significantly compared with the sham control group rats. The MDA level of the intestinal tissues was found to be significantly higher in the irradiated rats compared with the sham group rats. HO is an initial and rate-limiting enzyme. It oxdatively degrades heme into carbon monoxide, iron, and biliverdin, which subsequently is converted to bilirubin by biliverdin reductase. Bilirubin, as an antioxidant, provides cellular protection against free radical–mediated injury. CO regulates the generation of proinflammatory and anti-inflammatory cytokines. In addition, CO leads to vasodilatation and inhibition of platelet aggregation [8,9]. Two distinct isoforms of HO have been identified. The HO-2 isoform is constitutive and expressed physiologically. In contrast, the HO-1 isoform is induced strongly and rapidly by oxidative stress in pathologic conditions [11,12,23–25]. HO-1 induction is important in the response of tissue to oxidative stress and inflammation. Heat shock proteins, also known as HO-1, have been found to have a protective role in an experimental model [25]. Recent studies with HO-1 knock-out mice have shown that induction of HO-1 helps to ameliorate tissue injury or inflammation changes in a variety of experimental models [11,12,23]. Wang et al [26] reported that HO-1 was induced by hemin in colonic tissue damaged by experimental colitis. Prior administration with the HO activity inhibitor mesoporphyrin potentiated the colonic damage. Another study suggested that HO-1 induction by hemin is protective against ischemia-reperfusion injury [24]. In the present study, we showed that HO-1 expression was induced with glutamine and this improved intestinal damage produced by radiation. However, Zn-PP administration in irradiated rats worsened the intestinal damage by inhibiting HO-1 activitiy. During the inflammatory process, various cytokines are secreted into circulation. NF-B is activated by a wide
variety of agents, including hydrogen peroxide, ozone, reactive oxygen intermediates, interleukin-1, tumor necrosis factor-␣, bacteria, and viral transcriptions. Once activated, NF-B transcriptionally regulates many cellular genes implicated in early immune, acute phase, and inflammatory responses. The activation of NF-B depends on the cellular redox potential and the intracellular reduced glutathione/ oxidized glutathione ratio. The amount of activated NF-B correlates with the degree of mucosal inflammation [27– 29]. In the present study, NF-B positivity was strongest in the intestines of animals in irradiated rats. The expression was decreased in the glutamine-treated group. Apoptosis is an essential physiologic process required for maintenance of tissue homeostasis, insufficient or excessive cell death can contribute to disease [30,31]. Gastrointestinal diseases in which apoptosis has been implicated are inflammatory bowel disease, colon cancer, pancreatic cancer, acute pancreatitis, hepatitis, and radiation enteritis [32,33]. Dysregulation or inhibition of apoptosis appears to be important in cell proliferation, tissue hyperplasia, and malignant transformation of gastrointestinal epithelia. Although many factors are involved in the apoptotic program, caspases have been shown to play a major role in the transduction of apoptotic signals [31,32]. In the small intestine, spontaneous apoptosis can be seen. If an animal is exposed to irradiation, the levels of apoptosis in the small intestine increase markedly [31,34]. In the present study we found that abdominal irradiation caused an increase of caspase levels in the intestinal tissue whereas glutamine treatment significantly decreased these levels. In the Zn-PPtreatment group, intestinal caspase activity was found to be higher than in the group receiving only irradiation. Glutamine is the respiratory fuel for the mucosal cell of the intestine. Catabolic states such as major trauma, sepsis, major surgery, burns, transplantation, intestinal obstruction, radiotherapy, and chemotherapy are associated with low plasma levels of glutamine [13,14]. Glutamine improves trophism of enterocytes and colonocytes, enhances the immunologic barrier because of its trophic action on the immune system, serves as a substrate for glutathione, and is involved in antioxidant and scavenging actions on free radicals. In addition, glutamine effectively suppresses programmed cell death by decreasing death-receptor expression and caspase activities and increasing anti-apoptotic molecules [16,19]. Recent studies have suggested that pre-irradiation administration of a glutamine-enriched diet is beneficial to the intestinal mucosa after radiation exposure [1,5,6]. Uehara et al [35] reported that glutamine treatment significantly improves intestinal tissue injury in a rat model of endotoxemia. In addition, mesoporphyrin, known as an inhibitor of HO activity, completely abolished the protective effects of glutamine. In the present study, treatment with glutamine significantly decreased the MDA level increase in the intestinal tissue. In all irradiated rats, intestinal MPO activity was found to be increased significantly when compared with the sham group rats, whereas glutamine treatment decreased MPO increase. Glutamine treatment increased HO-1 expres-
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sion in the intestinal mucosal epithelium and prevented oxidative damage induced by irradiation. Inhibition of HO-1 activity by Zn-PP completely abolished the protective effects of glutamine. These results imply the reduction in mucosal damage is caused by the anti-inflammatory, anti-apoptotic, and HO-1 induction effects of glutamine. In conclusion, glutamine appears to have protective effects against the intestinal damage caused by abdominal radiation through the modification of the inflammatory response, the modification of apoptosis, and the induction of HO-1 activity.
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Scientific papers
The effect of heme oxygenase-1 induction by glutamine on radiation-induced intestinal damage: the effect of heme oxygenase-1 on radiation enteritis Murat Giris¸, M.S.C.a, Yes¸im Erbil, M.D.b,*, Serdar Öztezcan, M.D.a, Vakur Olgaç, Ph.D.c, Umut Barbaros, M.D.b, Ug˘ur Deveci, M.D.b, Banu Kirgiz, M.D.a, Müjdat Uysala, Gülçin Aykaç Tokera a
b
Department of Biochemistry, Istanbul University, Istanbul Medical Faculty, Capa, Istanbul, Turkey Department of General Surgery, Istanbul University, Istanbul Medical Faculty, 34340 Capa, Istanbul, Turkey c Department of Pathology, Istanbul University, Istanbul Medical Faculty, Capa, Istanbul, Turkey Manuscript received May 20, 2005; revised manuscript November 3, 2005
Abstract Background: Radiation enteritis is a significant clinical problem in patients receiving ionizing radiation directed at the abdomen or pelvis. The small intestine is the most radiosensitive gastrointestinal organ. Myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels of the small intestine were measured to determine the oxidative damage caused by radiation. In addition, caspase-3 activity of the small intestine was measured to define the degree of apoptosis. The present study was undertaken to investigate the effect of glutamine administration on heme oxygenase-1 (HO-1) expression of the radiation enteritis model. Methods: Rats received 1 g/kg/d glutamine (HO-1–inducer) for 7 days before irradiation and continued for 3 days after irradiation. Zn-prothoporphyin (Zn-PP) 40 mol/kg was delivered subcutaneously for 1 day before irradiation. Intestinal MPO activities and MDA levels are indicators of oxidative damage, whereas caspase-3 activities show the degree of apoptosis of the small intestine. At histopathologic examination, terminal ileum tissue was analyzed for morphologic changes. Also, the nuclear factor- (NF-) expression level of the terminal ileum was determined with immunohistochemisty methods to show the mucosal inflammatory process. Results: Irradiation significantly increased the intestinal MPO and caspase-3 activities, MDA levels, and HO-1 expression in comparison with the sham group. Glutamine treatment was associated with increased HO-1 expression, decreased MPO activity, caspase-3 activity, and MDA levels. Inhibition of HO-1 activity by Zn-PP completely eliminated the protective effects of glutamine. Histopathologic examination showed that the intestinal mucosal structure was preserved in the glutamine-treated group. In the irradiation group, NF-B overexpression was detected. NF-B positivity was strongest in the intestine of animals in the radiation alone group and the Zn-PP–treated irradiation group. Conclusions: Glutamine appears to have protective effects against radiation-induced intestinal damage. This protective effect is mediated in part by the induction of HO-1 activity because inhibition of Zn-PP resulted in the complete abolishment of the protective effect of glutamine. © 2006 Excerpta Medica Inc. All rights reserved. Keywords: Heme oxygenase-1; Glutamine; Zn-prothoporphyin; Irradiation; Apoptosis; NF-B
Radiation enteritis occurs during radiotherapy for many intraabdominal and pelvic cancers such as cervical, endometrial, ovarian, bladder, prostate, and rectal [1,2]. Abdominal irradiation causes mucosal damage in the gastrointestinal epithelium with activated inflammatory cells. The mucosal damage was described as destruction of crypt cells, a decrease in villous
* Corresponding author. Tel.: ⫹90-212-5331784; fax: ⫹90-212-6319771. E-mail address: [email protected]
height, and a decrease in the number, ulceration, and necrosis of the gastrointestinal epithelium [2– 4]. Although the pathogenesis of radiation enteritis is not clear, it is presumed to be an inflammatory process in which various mediators such as eicosanoids, cytokines, and reactive oxygen metabolites take place [4 –7]. Heme oxygenase (HO) is an enzyme that catalyzes the first and rate-limiting step in heme degradation, which yields the byproducts carbon monoxide (CO), free iron, and biliverdin, all of which possess free radical scavenging
0002-9610/06/$ – see front matter © 2006 Excerpta Medica Inc. All rights reserved. doi:10.1016/j.amjsurg.2005.11.004
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properties. HO-1 induction has been considered to protect cells against oxidative injuries [8 –12]. Glutamine is the respiratory fuel for the mucosal cell of the intestine. During pathologic conditions, the consumption of glutamine increases. In addition, glutamine has been shown to be beneficial in the prevention of mucosal integrity [13–16]. The mechanism of protection by glutamine remains unclear. In the present study we investigated whether HO-1 induction by glutamine could protect radiation-induced damage in the intestinal tissues by examining intestinal myeloperoxidase (MPO) activities and malondialdehyde (MDA) levels as indicators of oxidative damage and caspase-3 activitiy as an apoptosis marker. At histopathologic examination, the terminal ileum tissue was analyzed for morphologic changes. Also, the nuclear factor (NF-) expression level of the terminal ileum was determined with immunohistochemical methods to show the mucosal inflammatory process.
Materials and Methods Animals Fifty-six male Wistar-albino rats weighing 250 to 300 g (Istanbul University, Institute of Experimental Medicine and Research, Istanbul, Turkey) were used in the study. All animals were housed in wire-mesh– bottomed cages in a 12 hour light-dark cycle. Rats were kept in a room at a constant temperature of 22°C ⫾ 2°C and were fed a standard chow diet and water. The study was approved by the Ethics Committee of Istanbul University, Istanbul Medical School. Study design The rats were divided into 8 groups. Group 1 (abdominal irradiation [RT]; n ⫽ 8): abdominal irradiation alone; group 2 (RT/glutamine [Glu]; n ⫽ 8): abdominal irradiation and glutamine (Sigma, St. Louis, MO) 1 g/kg/d intragastric gavage for 7 days before irradiation and 3 days after irradiation; group 3 (RT/Zn-PP; n ⫽ 8): abdominal irradiation and zinc-prothoporphyin (Zn-PP; Sigma) 40 mmol/kg subcutaneously 1 day before radiation; group 4 (RT/Glu/Zn-PP; n ⫽ 8): abdominal irradiation and glutamine and Zn-PP; group 5 (Glu; n ⫽ 6): glutamine alone 1 g/kg/d intragastric gavage for 10 days; group 6 (Zn-PP; n ⫽ 6): Zn-PP alone 40 mmol/kg subcutaneously once; group 7 (Glu/Zn-PP; n ⫽ 6): Zn-PP and glutamine alone; group 8 (sham control group; n ⫽ 6): isotonic saline solution 1 cc/rat intragastric for 10 days. Glutamine was administered 7 days before radiation and 3 days after irradiation. All animals were irradiated on day 7 and killed on day 10. Zn-PP was dissolved in .1 mL of .5 mol/L NaOH; normal saline was added until the drug preparation equaled .5 mL and the pH was adjusted to 7.4,
and was stored in the dark to prevent photodegradation. Zn-PP was administered 1 day before radiation. Irradiation consisted of a single dose of 1,000 cGy (source to skin distance, 80 cm) to the abdominal region of the animals anesthetized with sodium thiopental 40 mg/kg intraperitoneally. Three days after irradiation, all animals were killed with a lethal dose of sodium thiopental. The small intestine then was removed and washed with ice-cold isotonic saline solution. Samples of the terminal ileum were fixed in buffered formalin for histopathologic examination. The rest of the tissue was stored at ⫺80°C until it was processed for biochemical analysis. MPO activity Intestinal MPO activity was assayed by the o-dianisidine method [17]. Tissue samples were suspended in 50 mmol/L, pH 6.0, potassium phosphate buffer containing .5% hexadecyltrimethylammonium bromide and homogenized. A sample of homogenate was sonicated for 20 seconds, freezethawed 3 times, sonicated again for 10 seconds, and centrifuged at 15,000 ⫻ g for 10 minutes at 4°C. The reaction was started by mixing and incubating the 100 L of the supernatant at 25°C with a solution composed of 2.810 L of 50 mmol/L potassium phosphate buffer, pH 6.0, 30 L of 20 mg/mL o-dianisidine dihydrochloride, and 30 L of 20 mmol/L H2O2. After 10 minutes the reaction was terminated by the addition of 30 L of 2% sodium azide. The change in absorbance was read at 460 nm and the results were calculated by using the molar absorptivity coefficient of oxidized o-dianisidine (1.0062 ⫻ 104 mol/L⫺1 cm⫺1). MPO activity (1 unit) was expressed as the amount of enzyme necessary for the degradation of 1 mol of H2O2/min/100 mg tissue at 25°C. MDA assays The levels of MDA in the tissues were measured to assess lipid peroxidation. Samples of the small intestine were homogenized with ice-cold 150 nmol/L potassium chloride for the determination of MDA levels. MDA levels were measured spectrophotometrically. Results are expressed as nmol of MDA per gram of tissue [18]. Caspase activity Caspase-3 activity was measured by quantifying the cleavage of acetyl-Asp-Glu-Val-Asp p-nitroaniline with a colorimetric caspase-3 assay kit (Sigma). P-nitroaniline released by the enzymatic hydrolysis of acetyl-Asp-Glu-ValAsp p-nitroaniline was calculated from a p-nitroaniline standard carve and the molar absorptivity coefficient of p-NA at 405 nm (10,500 mol/L⫺1 cm⫺1). The supernatant obtained by the centrifugation of 10% tissue homogenate at 15,000 ⫻ g for 5 minutes at 4°C was used as an enzyme source and
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incubated with and without caspase-3 inhibitor, Ac-DEVCHO, according to the assay protocol of the kit. Caspase activity was calculated by substracting the absorbance measured in the presence of substrate plus inhibitor from the absorbance observed by incubating with substrate alone for 20 minutes at 37°C. The activity of the enzyme was expressed as p-nitroaniline liberated per mg protein per minute. The protein concentration of the enzyme source was measured by using bicinchoninic acid [19]. Western blot analysis for HO-1 Western blot analysis was performed with modifications as previously described [8]. Briefly, livers were homogenized in the 5 volume lysis buffer containing 10 mmol/L Tris-HCl (pH 8), 140 mmol/L NaCl, .5 mmol/L ethylenediaminetetraacetic acid, 1% TritonX-100, 1% deoxycholate, .1% sodium dodecyl sulfate, .5 mmol/L phenylmethylsulfonyl fluoride, 1 g/L leupeptin, and 1 g/L aprotinin. The homogenate was centrifuged at 10,000 ⫻ g for 15 minutes at 4°C. The supernatant was centrifuged at 105,000 ⫻ g for 1 hour at 4°C. The precipitated microsome was dissolved in the earlier-described Tris-HCl solution. Equal amounts of protein (100 g) were loaded onto 12% sodium dodecyl sulfate–polyacrylamide gels and blotted onto the polyvinylidine difluoride membrane (Millipore, MA). Nonspecific binding sites were blocked with Trisbuffered saline–Tween 20 containing 5% nonfat dry milk overnight at 4°C. Membranes were incubated in polyclonal antibody against rat HO-1 at 1:1,500 dilutions and horseradish-peroxidase– conjugated secondary antibody (goat anti-rabbit IgG; Stress Gen, Toronto, Canada) at 1:16,000 dilutions. The membrane-bound antibody was visualized with the enhanced chemiluminescence Western blotting detection system kit (Amersham, Pittsburgh, PA). Bands were quantitated using a densitometer (image analysis software, Vilber Lourmat Biotechnology, Marne-la-Vallée, France).
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hematoxylin. The staining intensity was defined in percentages and was given scores ranging from 1 to 4 (⬎75%, 4⫹; 50%–75%, 3⫹; 25%–50%, 2⫹; ⬍25%, 1⫹. Statistical analysis All data were expressed as mean ⫾ SD. The MannWhitney U test and analysis of variance were used for statistical analysis and a P value of less than .05 was considered significant (SPSS 11.0 for Windows, Chicago, IL). Results All rats developed radiation-induced diarrhea within 24 hours after abdominal radiation. There were no treatmentrelated mortalities. HO-1 protein expression Fig. 1 shows that treatment with glutamine in the irradiated rats significantly induced HO-1 expression when compared to sham control rats (49 ⫾ 8 vs. 15 ⫾ 4). Irradiation similarly induced HO-1 expression (30 ⫾ 5). HO-1 expression seen in the group given Zn-PP was higher than in the control group. This was believed to be the result of an increase in HO-1 expression on a molecular basis to compensate for the decrease in HO-1 activity. The increase in MPO and MDA levels also is proof of a Zn-PP–suppressor effect on HO-1 activity. MPO activity In all irradiated rats, intestinal MPO activity was found to be increased significantly compared with the sham con-
Histopathology The terminal ileum tissue was analyzed for morphologic changes. The changes in the terminal ileum mucosa were evaluated for mean villous height and the number of villous per unit by micrometer-adapted light microscope. NF-B expression All specimens were fixed in 10% buffered formalin. Paraffin blocks prepared from routinely processed specimens were cut into 5 slices and were deparaffinized. Antigen retrieval was performed after this process. After microwave incubation of the peroxyblock, followed by ultra V block procedure, primary antibodies (NF-B neomarkers anti-rabbit P50AB-2) were applied. After this process, biotinlated secondary antibody, streptovidin peroxidase, and substrat-cromogen (amino ethyl carbasole) solution was applied, respectively. Nuclear staining was performed with
Fig. 1. Intestinal HO-1 protein expression in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .05; a and c is P ⬍ .05; a and d is P ⬍ .001; b and d is P ⬍ .001; c and d is P ⬍ .001.
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Fig. 2. Intestinal MPO activity in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .001; a and c is P ⬍ .001; b and c is P ⬍ .001.
trol group rats (P ⬍ .001, 6.9 ⫾ 1.2 Ug/tissue vs. 2.1 ⫾ .4 Ug/tissue). Intestinal MPO activity was significantly lower in the glutamine-treated irradiation group compared with the group receiving only irradiation (P ⬍ .001, 2.3 ⫾ .2 Ug/tissue vs. 6.9 ⫾ 1.2 Ug/tissue). In the Zn-PP–treated irradiation group, intestinal MPO activity (11.9 ⫾ 2.2 Ug/ tissue) was found to be increased significantly compared with all other groups including the group receiving only irradiation (P ⬍ .001). However, Zn-PP alone did not change MPO activity compared with the other control groups (Fig. 2). MDA levels The MDA level of the intestinal tissues was found to be significantly higher in the irradiated rats compared with the sham group rats (1.9 ⫾ .2 nmol MDA/mg protein vs. .9 ⫾ .1 nmol MDA/mg protein, P ⬍ .001). Treatment with glutamine significantly decreased the MDA levels compared with the group receiving only irradiation (.8 ⫾ .2 nmol MDA/mg protein vs. 1.9 ⫾ .2 nmol MDA/mg protein, P ⬍ .001). In the Zn-PP–treated irradiation group, intestinal MDA activity was found to be decreased significantly compared with the group receiving only irradiation (1.7 ⫾ .1 nmol MDA/mg protein vs. 1.9 ⫾ .2 nmol MDA/mg protein), but increased significantly compared with the other groups (P ⬍ .001). However, Zn-PP alone did not change MDA activity compared with the other control groups (Fig. 3).
Fig. 3. Intestinal MDA levels in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .001; a and c is P ⬍ .001; b and c is P ⬍ .001.
only irradiation (4.3 ⫾ 1.2 nmol mg protein/min vs. 13.2 ⫾ 1.9 nmol mg protein/min, P ⬍ .05). In the Zn-PP–treated irradiation group, intestinal caspase-3 activity (16.9 ⫾ 3.2 nmol mg protein/min) was found to be increased significantly compared with all other groups including the group receiving only irradiation (P ⬍ .001). However, Zn-PP alone did not change caspase-3 activity compared with the other control groups (Fig. 4). Histopathologic assessment Ileal villous number and height were found to be significantly lower in irradiated rats compared with sham group rats (4.6 ⫾ .6 and .14 ⫾ .01 mm vs. 9.1 ⫾ .9 and .48 ⫾ .05 mm, respectively, P ⬍ .001). Glutamine pretreatment before irradiation significantly prevented a decrease in villous number and height (7.9 ⫾ .5 and .33 ⫾ .01 mm,
Caspase-3 activity The caspase-3 activity of the intestinal tissues was found to be significantly higher in the irradiated rats compared with the sham group rats (13.2 ⫾ 1.9 nmol mg protein/min vs. 3.4 ⫾ .2 nmol mg protein/min, P ⬍ .001). Glutamine administration significantly decreased the caspase-3 activity of the intestinal tissues compared with the group receiving
Fig. 4. Intestinal caspase-3 activitiy in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .001; a and c is P ⬍ .05; a and d is P ⬍ .001; b and d is P ⬍ .05; c and d is P ⬍ .001.
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Fig. 7. Mean ⫾ SD ratio of NF-B expression of the rat intestines in all groups. Letters signify different values according to analysis of variance testing. The significance between a and b is P ⬍ .001; a and c is P ⬍ .05; a and d is P ⬍ .001; b and c is P ⬍ .05; b and d is P ⬍ .05.
respectively). In the Zn-PP–treated irradiation group, villous number and height were found to be decreased significantly (3.6 ⫾ .8 and .13 ⫾ .03 mm, respectively) compared with all groups other than the group receiving only irradiation (P ⬍ .001) (Figs. 5 and 6).
Fig. 5. Mean ⫾ SD of (A) number of villi and (B) villous height in all groups. Letters signify different values according to analysis of variance testing. (A) The significance between a and b is P ⬍ .001; a and c is P ⬍ .05; a and d is P ⬍ .05; b and c is P ⬍ .05; b and d is P ⬍ .05. (B) The significance between a and b is P ⬍ .05; a and c is P ⬍ .05; b and c is P ⬍ .05.
NF-B expression NF-B positivity was strongest in the intestines of animals in the group receiving only irradiation and the Zn-PP– treated irradiation group. NF-B expression was decreased in the glutamine-treated group (Figs. 7 and 8). Comments The results of the present study suggest that oxidative damage, apoptosis, and HO-1 activity contribute to the
Fig. 6. (A) Distal ileum from irradiation group. There is villous shortening and the villous epithelium has been damaged completely, hematoxylin-eosin ⫻ 40. (B) Distal ileum from glutamine-treated irradiation group. There is pronounced epithelial damage that has been preserved partly, hematoxylin-eosin ⫻ 40. (C) Distal ileum from the Zn-PP–treated irradiation group. The mucosal epithelium has been damaged completely, hematoxylin-eosin ⫻ 40. (D) Distal ileum from the glutamine- and the Zn-PP–treated irradiation group. The mucosal epithelium has been preserved partly, hematoxylin-eosin ⫻ 40. (E) Distal ileum from glutaminealone group. (F) Distal ileum from Zn-PP alone group. (G) Distal ileum from glutamine and Zn-PP group. (H) Distal ileum from control group. Last four groups are seen normal intestinal histologic findings, hematoxyline-eosin ⫻ 100.
Fig. 8. (A) Nuclear kappa factor-B (NF-B) overexpression in the intestine in the irradiation group, NFB ⫻ 40. (B) Decreased NF-B expression in the intestine in the glutamine-treated irradiation group, NFB ⫻ 40. (C) NF-B overexpression in the intestine in the Zn-PP–treated irradiation group, NFB ⫻ 40. (D) Decreased NF-B expression in the intestine in the glutamine- and Zn-PP–treated irradiation group, NFB ⫻ 100. (E) Distal ileum from glutamine-alone group. (F) Distal ileum from Zn-PP alone group. (G) Distal ileum from glutamine and Zn-PP group. (H) Distal ileum from control group. In the last four groups, NF-B expression was not seen. Normal intestinal histologic findings are seen, NF-B ⫻ 100.
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development of radiation enteritis after abdominal radiation, as assessed by increased lipid peroxidation, MPO activity, caspase-3 levels, and HO-1 induction in the intestinal tissue. Our results also show that glutamine protects against radiation-induced intestinal damage. The protective effect of glutamine is related in part to site-specific HO-1–inducing activity in the intestinal mucosa, which leads to the reduction of tissue inflammation and the suppression of apoptosis. Radiation enteritis occurs as a response to abdominal radiation, which can cause mucosal damage in the gastrointestinal mucosal epithelium. The small intestine is one of the most radiosensitive organs in the abdomen, and in the present study we have shown a significant decrease in the number and height of the intestinal villi of the small intestine by histopathologic examination in irradiated animals [5–7]. This finding is consistent with radiation-induced damage and inflammation. Radiation also can activate inflammatory cells, leading to the synthesis and release of certain cytokines, inflammatory mediators, and reactive oxygen metabolites [20 –22]. In our study, in all irradiated rats, the intestinal MPO activity was found to be increased significantly compared with the sham control group rats. The MDA level of the intestinal tissues was found to be significantly higher in the irradiated rats compared with the sham group rats. HO is an initial and rate-limiting enzyme. It oxdatively degrades heme into carbon monoxide, iron, and biliverdin, which subsequently is converted to bilirubin by biliverdin reductase. Bilirubin, as an antioxidant, provides cellular protection against free radical–mediated injury. CO regulates the generation of proinflammatory and anti-inflammatory cytokines. In addition, CO leads to vasodilatation and inhibition of platelet aggregation [8,9]. Two distinct isoforms of HO have been identified. The HO-2 isoform is constitutive and expressed physiologically. In contrast, the HO-1 isoform is induced strongly and rapidly by oxidative stress in pathologic conditions [11,12,23–25]. HO-1 induction is important in the response of tissue to oxidative stress and inflammation. Heat shock proteins, also known as HO-1, have been found to have a protective role in an experimental model [25]. Recent studies with HO-1 knock-out mice have shown that induction of HO-1 helps to ameliorate tissue injury or inflammation changes in a variety of experimental models [11,12,23]. Wang et al [26] reported that HO-1 was induced by hemin in colonic tissue damaged by experimental colitis. Prior administration with the HO activity inhibitor mesoporphyrin potentiated the colonic damage. Another study suggested that HO-1 induction by hemin is protective against ischemia-reperfusion injury [24]. In the present study, we showed that HO-1 expression was induced with glutamine and this improved intestinal damage produced by radiation. However, Zn-PP administration in irradiated rats worsened the intestinal damage by inhibiting HO-1 activitiy. During the inflammatory process, various cytokines are secreted into circulation. NF-B is activated by a wide
variety of agents, including hydrogen peroxide, ozone, reactive oxygen intermediates, interleukin-1, tumor necrosis factor-␣, bacteria, and viral transcriptions. Once activated, NF-B transcriptionally regulates many cellular genes implicated in early immune, acute phase, and inflammatory responses. The activation of NF-B depends on the cellular redox potential and the intracellular reduced glutathione/ oxidized glutathione ratio. The amount of activated NF-B correlates with the degree of mucosal inflammation [27– 29]. In the present study, NF-B positivity was strongest in the intestines of animals in irradiated rats. The expression was decreased in the glutamine-treated group. Apoptosis is an essential physiologic process required for maintenance of tissue homeostasis, insufficient or excessive cell death can contribute to disease [30,31]. Gastrointestinal diseases in which apoptosis has been implicated are inflammatory bowel disease, colon cancer, pancreatic cancer, acute pancreatitis, hepatitis, and radiation enteritis [32,33]. Dysregulation or inhibition of apoptosis appears to be important in cell proliferation, tissue hyperplasia, and malignant transformation of gastrointestinal epithelia. Although many factors are involved in the apoptotic program, caspases have been shown to play a major role in the transduction of apoptotic signals [31,32]. In the small intestine, spontaneous apoptosis can be seen. If an animal is exposed to irradiation, the levels of apoptosis in the small intestine increase markedly [31,34]. In the present study we found that abdominal irradiation caused an increase of caspase levels in the intestinal tissue whereas glutamine treatment significantly decreased these levels. In the Zn-PPtreatment group, intestinal caspase activity was found to be higher than in the group receiving only irradiation. Glutamine is the respiratory fuel for the mucosal cell of the intestine. Catabolic states such as major trauma, sepsis, major surgery, burns, transplantation, intestinal obstruction, radiotherapy, and chemotherapy are associated with low plasma levels of glutamine [13,14]. Glutamine improves trophism of enterocytes and colonocytes, enhances the immunologic barrier because of its trophic action on the immune system, serves as a substrate for glutathione, and is involved in antioxidant and scavenging actions on free radicals. In addition, glutamine effectively suppresses programmed cell death by decreasing death-receptor expression and caspase activities and increasing anti-apoptotic molecules [16,19]. Recent studies have suggested that pre-irradiation administration of a glutamine-enriched diet is beneficial to the intestinal mucosa after radiation exposure [1,5,6]. Uehara et al [35] reported that glutamine treatment significantly improves intestinal tissue injury in a rat model of endotoxemia. In addition, mesoporphyrin, known as an inhibitor of HO activity, completely abolished the protective effects of glutamine. In the present study, treatment with glutamine significantly decreased the MDA level increase in the intestinal tissue. In all irradiated rats, intestinal MPO activity was found to be increased significantly when compared with the sham group rats, whereas glutamine treatment decreased MPO increase. Glutamine treatment increased HO-1 expres-
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sion in the intestinal mucosal epithelium and prevented oxidative damage induced by irradiation. Inhibition of HO-1 activity by Zn-PP completely abolished the protective effects of glutamine. These results imply the reduction in mucosal damage is caused by the anti-inflammatory, anti-apoptotic, and HO-1 induction effects of glutamine. In conclusion, glutamine appears to have protective effects against the intestinal damage caused by abdominal radiation through the modification of the inflammatory response, the modification of apoptosis, and the induction of HO-1 activity.
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