Small Intestinal Mucositis in the Rat

©2005L AN DE S B IO SC I EN CE .D ONO T D I S TR I B U T E.[Cancer Biology &Therapy 5:1, 34-38, January 2006]; ©2005 Landes BioscienceJulie M. Clarke 1,5Nicole C. Pelton 2,3Balazs H. Bajka 1,3Gordon S. Howarth 1,2,3,4,5,6,7Leanna C. Read 1,5Ross N. Butler 2,3,4,51Child Health Research Institute; 2Centre for Paediatric and AdolescentGastroenterology, Children, Youth and Women’s Health Service; Departments of3Physiology and 4Paediatrics; University of Adelaide5Cooperative Research Centre for Tissue Growth and Repair; 6School of Pharmaceutical, Molecular and Biomedical Sciences; University of South Australia 7School of Biological Sciences; Flinders University; Adelaide, South Australia,Australia*Correspondence to: Ross N. Butler; Children, Youth and Women’s Health Service; Centre for Paediatric and Adolescent Gastroenterology; 72 King William Road; North Adelaide, South Australia 5006 Australia; Tel.: +61.8.8161.6805;Fax: +61.8.8161.6088; Email: ross.butler@.auReceived 09/01/05; Accepted 10/12/05Previously published online as a Cancer Biology & Therapy E-publication:/journals/cbt/abstract.php?id=2235KEY WORDS13C-sucrose, breath test, sucrase, mucositis,methotrexate, folinate, ratABBREVIATIONSDAR Dark Agouti ratH&E Hematoxylin and eosin MPO Myeloperoxidase MTX MethotrexateSBT Sucrose breath test AUC Area under curveACKNOWLEDGEMENTSThe authors wish to thank Helen Bougesis and Ben Edwards for conducting the animal trials,Caroline Payne for biochemical analyses and Katie Inglis for assistance with the preparation of the manuscript. The Co-operative Research Centre for Tissue Growth and Repair and Nestec Ltd are also acknowledged and thanked for financial support of this study.Research Paper Use of the 13C-Sucrose Breath Test to Assess Chemotherapy-Induced Small Intestinal Mucositis in the RatABSTRACTMucositis is a debilitating side-effect of chemotherapy which affects the mucosa of the gastrointestinal tract, particularly the small intestine. Currently there are no simple, non-invasive methods to detect and monitor small intestinal function and the severity of mucosal damage. Activity of the brush-border enzyme sucrase provides an indicator of small intes-tinal absorptive function that remains relatively constant throughout life. Measuring 13CO 2levels in expired breath following ingestion of 13C-sucrose is a non-invasive marker of total intestinal sucrase activity. We evaluated the sucrose breath test (SBT) as an indicator of small intestinal injury and dysfunction, utilizing a rat model of chemotherapy-induced mucositis. SBT results reflected the time-course of damage and repair after methotrexate (MTX) treatment, with damage most severe 72 h after chemotherapy, and repair commenc-ing after 96 h. SBT results correlated significantly with jejunal sucrase activity determined biochemically (r 2 = 0.89; p < 0.005). Moreover, calcium folinate ingested prior to chemotherapy totally prevented damage to the small intestinal mucosa induced by MTX,as assessed by the SBT in concert with structural, and biochemical indices. The SBT provides a simple, non-invasive, integrated measure of small intestinal damage and function.The SBT holds significant potential to monitor small intestinal function in cancer patients undergoing chemotherapy. This technique possesses further applicability to the screening of newly-developed agents for potential gastrointestinal toxicity including the development of new therapies targeted at minimising or preventing the onset of chemotherapy-induced mucositis.INTRODUCTIONCurrently, there is no simple, non-invasive and reliable method to assess small intestinal function and detect the development of mucosal damage in patients undergoing chemotherapy. Such a technique would be valuable in both clinical management and research studies to assess the efficacy of therapies aimed at reducing gut mucositis.Mucositis is a serious and common side effect of chemotherapy, with approximately 60% of all patients receiving high dose treatment developing the condition.1Moreover,paediatric patients in whom epithelial cell turnover is more rapid, are at greater risk of developing intestinal mucositis.2Indeed, mucositis remains the major dose-limiting side-effect of chemotherapy as traditional treatment modalities are often ineffective and limited to symptomatic relief. Mucositis results from the direct inhibitory effects of chemo-and radiation-therapy on DNA replication and mucosal cell proliferation,3resulting in apoptosis and hypo-proliferation in intestinal crypts, villous atrophy, collagen break-down, ulceration and abnormal intestinal permeability in cancer patients. The ulcerative lesions are painful, forming sites for secondary infection that may be potentially fatal in neutropenic patients.4The development of life-threatening mucositis following high doses of specific chemotherapeutic agents, such as MTX, has necessitated the intravenous administration of folinate (Leucovorin®) 24–36 h after high dose MTX treatment in order to “rescue” patients from MTX toxicity.3In the healthy small intestine, sucrose is digested by the brush-border enzyme sucrase,5the products are then metabolised in the liver and expired in the breath. The SBT involves the ingestion of a naturally enriched 13C-substrate (sucrose), and the collection of timed breath samples for the measurement of expired 13CO 2. Sucrase is a relatively stable brush-border enzyme,6with similar levels of activity recorded throughout life once the small intestine has matured. Mucositis-induced intestinal damage diminishes sucrase activity as a consequence of villus atrophy and hence, a reduced absorptive surface area.7Reduced levels of expired 13CO 2therefore reflect both the extent of small intestinal damage and the loss of absorptive and digestive capacity.Recently, we reported applicability of the SBT to the detection of intestinal mucositis induced by MTX in rats.8The primary aim of the present study was to further application of the SBT to evaluate its ability to detect attenuation of MTX-induced small intestinal toxicity following administration of oral folinate (Leucovorin®). MATERIALS AND METHODSAll animal experimentation was approved by the Animal Ethics Committee of the Children, Youth and Women’s Health Service, Adelaide and complied with the Australian Code of Practice for the Use of Animals in Research and T eaching (1997).Time course of intestinal mucositis in the dark agouti rat.This study investigated the effects of MTX chemotherapy on the function and structure of the small intestine during the processes of damage and repair in 24 female Dark Agouti rats (DAR), initial body weight 115.8 ±1.0 g (Institute of Medical and Veterinary Science, Gilles Plains, Adelaide, South Australia). The chemotherapy treatment has been previously described.9In brief, the procedure involved the intramuscular injection of 1.5 mg/kg MTX (Pharmacia and Upjohn Pty Ltd, Bentley WA, Australia) at times 0 and 24 h of the experimental period. MTX is a widely used antimetabolite which disrupts normal DNA synthesis in the S-phase of the cell cycle by inhibition of the enzymes dihydrofolate reductase and thymidylate synthase,10resulting in myelosuppression and subsequent development of mucositis.11 Rats were maintained in T ecniplast®metabolism cages throughout the experimental period, and given ad libitim access to a semi-synthetic diet12 and fresh water. Food intakes and body weights were measured daily. The SBT procedure was undertaken prior to MTX treatment, and again imme-diately prior to kill. Rats were anaesthetised using halothane (Fluothane®, Zeneca Macclesfield Cheshire, UK), cardiac blood samples collected, and killed by cervical dislocation 30, 48, 72, 96 and 120 h after the first MTX injection (n = 4/group). A group of non-MTX treated control animals was killed 120 h after the first MTX injection. Samples of jejunum and ileum were collected into liquid nitrogen and stored at -80˚C for measurements of intestinal sucrase and myeloperoxidase (MPO) activity, and into 10% buffered formalin for histological evaluation and qualitative scoring of intes-tinal damage.13The semi-quantitative histological scoring procedure involved the blinded assessment of intestinal tissues (increasing in severity from 0–3) for eleven independent criteria including aspects of villus and crypt integrity in addition to effects on lymphoid cell infiltration, oedema and wall thickening. These parameters have been described comprehensively previously.13Serum and blood were submitted to Veterinary Pathology Services Pty Ltd, Adelaide, South Australia, where a Coulter counter T890 using a veterinary mode was used to undertake routine haematological measurements. Differential blood counts were undertaken manually.Folinate administration.Calcium folinate (Leucovorin®, David Bull Laboratories, Mulgrave North, Victoria Australia), a metabolite and active form of folic acid, is a MTX antagonist used clinically to “rescue” normal cells from MTX damage, and hence, treat patients with symptoms of MTX-induced gastrointestinal toxicity. Sixteen female DAR (initial weight 151.8±1.2 g) were injected intramuscularly with MTX (1.5 mg/kg) at times 0 and 24 h. Calcium folinate was added to the drinking water of eight rats 2 h prior to and during MTX treatment, for a total of 48 h (MTX+F). The calcium folinate dose approximated that used clinically in humans (10 mg/m2) calculated using allometric scaling.14Eight rats were injected with MTX but received no folinate treatment (MTX-F), and four rats received no treatments (control). Four rats from each treatment group were killed at 72 h, and the remaining four at 120 h after the initial MTX injection. The SBT was performed before chemotherapy, and at 52 and 100 h after the initial MTX injection. In the folinate study the rats were fasted for 3 h and the SBT undertaken from 11am; whilst in the first study the rats were fasted overnight. The difference in fasting schedules between the studies did not significantly affect baseline levels of breath 13CO2production (data not shown).Sucrose breath test.Rats were individually placed in perspex chambers to acclimatise for 15 minutes, and two baseline breath samples were collected (T0). Breath samples were collected in duplicate by syringe from two-way taps fitted to the collection chambers; and transferred to 10 ml evacuated glass tubes (Exetainer, Labco Limited, High Wycombe, England) as described previously.15At T0rats were gavaged with 1.0 ml of a solution containing 1 g/ml of selectively enriched 13C-sucrose (AnalaR BDH, MERCK, Pty Ltd, Victoria, Australia) and breath samples collected at 30-minute intervals. Breath samples were also collected for measurement of hydrogen (H2), to ensure breath 13CO2was not derived from colonic bacterial fermentation.16 On the basis of breath H2production data from the first study, the time-point of 90 minutes after gavage was selected as the appropriate time period during which breath 13CO2was solely derived from small intestinal enzyme activity.Analytical techniques.Breath samples were analysed for 13CO2by isotope ratio mass spectrometry (IRMS; Europa Scientific ABCA 20/20 Crewe, UK). Results were calculated as the change in breath 13CO2levels from baseline for each time point of breath collection throughout the period of interval sampling. The area under the curve (AUC) at 90 minutes after 13C-sucrose gavage was calculated using the trapezoidal rule:AUC0-t= Σ[(DOB t1+ DOB t2)/2] x (t2 -t1)Sucrase activity was measured in tissue by a modification of the glucose oxidase method for estimation of glucose concentration.17Based on the lowest standard used in the assay, the lowest detectable level was taken as 2.5 nmol glucose/min/cm.Myeloperoxidase (MPO) is an enzyme found in high levels in the pri-mary granules of neutrophils, and is considered a marker for tissue activated neutrophil content and hence, an indicator of the degree of tissue inflam-mation. Small intestinal MPO tissue levels were measured by a modification18 of a previously published technique.19Tissue samples were suspended in 0.5% hexadecyltrimethyl ammonium bromide (Sigma Chemical Co., St Louis, Mo., USA) pH 6.0 and homogenized for 30 s. Homogenates were made up to a final concentration of 50 mg tissue per ml of buffer. Samples were then frozen and thawed and centrifuged at 13000 g for 2 min. The MPO activity in the supernatant was measured spectrophotometrically. Aliquots were sampled onto a 96 well plate with o’dianisidine (Sigma Chemical Co., St Louis, Mo., USA) and 0.0005% hydrogen peroxide (BDH. Poole, Dorset, England), and the change in absorbance at 450nm measured using a microtitre plate scanner.Villus heights were measured in formalin fixed intestinal tissues that had been embedded in paraffin wax, sectioned at 4 µm thickness and stained with Harris haematoxylin and eosin. Sections were analysed by light microscopy (Olympus BH2, Olympus Optical Tokyo, Japan). Images were digitised for computer-aided analysis via a digital camera (Sony Hyper HAD Digital video camera model SSC-DC50P, Aomori, Japan) and analysed using Image-Pro Plus 4.0 software (Media Cybernetics, Silver Springs, Md., USA).13Statistical analyses.GraphPad Prism version 4.0 () was used for statistical analyses and for generation of graphs. Normally distributed data was compared using one-way analyses of variance using T ukey’s post-hoc tests, and histological scores were compared using non-parametric Kruskal-Wallis tests with Dunns post tests. Pearson’s product-moment correlation was used to compare breath 13CO2 levels (AUC) and the in vitro sucrase activity of the small intestine. For statistical purposes data from control animals in the folinate study which were killed at 72 h and 120 h were pooled, as were the prechemotherapy 13CO2levels of MTX-treated rats and MTX-treated rats receiving folinate. With the exception of the histological scores, (expressed as medians with ranges), all data are expressed as mean ±SEM with statistical significance indicated when p < 0.05.RESULTSTime course of small intestinal mucositis in the DAR.The plasma white cell counts were reduced from a control mean of 7.4 ±1.3 x 109/L to a mean of 4.3 ±1.2 x 109/L at 30 h after initial chemotherapy, implying the rats were moderately immunosuppressed by MTX treatment. MTX injec-tions resulted in significant reductions in food intake (12.4 ±0.27 g to 4.7 g ±2.3) and body weight (123.3 ±1.1 g to 117.2 ±4.5 g) from day 0 to 96 h respectively, and significantly increased the weights of the small intestine from a control value of 2.80 ±0.1 g to 4.56 ±0.23 g at 120 h post chemotherapy (p < 0.01). Medians and ranges of the histological severity scores are illustrated in Figure 1 MTX caused significant damage to the structure of the proximal jejunum; with damage most severe 72 h after the first MTX injection, and repair commencing by 120 h.Jejunal tissue sucrase activity declined rapidly from control levels of 452±71 to 21 ±11 nmol glucose/min/cm 72 h after chemotherapy. By 72 and 96 h after MTX treatment only two and one value respectively were above the detection limit of the assay. 120 h after MTX treatment jejunal sucrase activity increased to 39 ±14 nmol glucose/min/cm. Ileal tissue sucrase activ-ity also decreased after MTX treatment; from control levels of 82 ±5 nmol glucose/min/cm to 26 ±23 nmol glucose/min/cm at 72 h. Ileal levels increased rapidly and were restored to prechemotherapy levels of 90 ±7 nmol glucose/min/cm 120 h post chemotherapy. This result reflected the less severe chemotherapy-induced damage in the ileum compared with the jejunum.Breath 13CO2levels in MTX-treated rats were lower at 30, 48 and 72 h in comparison to baseline levels; whereas levels at the 96 and 120 h time-points were significantly higher than at 72 h. The AUC results were also significantly lower at the 48, 72, 96 and 120 h time-points compared with controls (Fig. 2). The mean jejunal tissue sucrase activities for all groups significantly correlated with mean breath 13CO2levels (r2= 0.89, p < 0.005).Effects of folinate on small intestinal function.Food intake of folinate-supplemented rats (MTX+F) was similar to that of non-MTX treated controls, whereas intakes of MTX-treated rats not given folinate (MTX-F) were signif-icantly lower at 48, 72 and 96 h (p < 0.05, (Fig. 3). These reductions in intake were reflected in differences in body weights (data not shown).Biochemical determinations of intestinal sucrase activity were significantly lower in MTX-F rats compared with controls (p < 0.05), whereas MTX+F rats were protected from the effects of chemotherapy (Fig. 4). Villus heights were also significantly lower in MTX-F treated rats at 72 h (331 ±17 µm) compared to controls (610 ±14 µm; p < 0.001) and the MTX+F treated group (531 ±32 µm; p < 0.001) supporting treatment with oral folinate as a protective agent for the small intestine.The AUC generated from 13CO2production (Fig. 5) was significantly lower in MTX-F treated rats compared to their MTX+F treated counterparts (p < 0.05), and the AUC results were also significantly lower at both 52 and 100 h post-chemotherapy compared to pre-MTX values (p < 0.05). Importantly, the mean jejunal tissue sucrase activities for all groups in the folinate study were significantly correlated with mean breath 13CO2levels (r2= 0.90, p < 0.005). The amount of 13C-labelled CO2, as measured by AUC, was significantly lower in MTX-F treated rats compared to MTX+F (Fig. 5). MPO levels of MTX-F treated rats were also significantly higher than MTX+F treated and control rats at 72 h (p < 0.01, < 0.001 respectively) (Fig. 6), but were decreased by 120 h after chemotherapy. DISCUSSIONThe severity of intestinal mucositis is a limiting factor in chemotherapy regimens20,21and, to date, the capability to predict the onset of mucositis and to monitor its severity has been hindered by the lack of a suitable non-invasive test to assess small intestinal function. This study has utilised a simple stable isotope breath test to detect damage and measure functional capacity of the small intestineFigure 1. Effects of two consecutive daily injections of MTX on the proximal jejunal histological architecture: Severity score.Figure 2. Area under the curve (AUC) of 13CO2production of rats 90 min-utes after sucrose gavage before and after treatment with MTX (n=4 except n=17 for control, mean ±SEM). *,**,*** significantly different to control (p < 0.05, < 0.01 and < 0.001).Figure 3. Food intakes (g/day) of rats administered oral folinate and injected with MTX (n=4, mean ±SEM; L MTX + folinate, M MTX no folinate; con-trol n=4 to 72 h, n=2 to 120 h;); *MTX + folinate and control significantly different to MTX no folinate group (p < 0.05); +MTX and folinate signifi-cantly different to MTX no folinate group (p < 0.05).in rodents following administration of MTX. In human studies the focus for predicting and assessing the severity and potential efficacy of anti-mucositis agents has relied on monitoring changes in the oral cavity.22-24When oral symptoms occur they present 7–10 days after chemotherapy.2,25,26However, small intestinal damage is likely to occur earlier since columnar epithelium is known to be more sus-ceptible to damage than squamous epithelium. Changes in the oral cavity may therefore not reflect the severity of damage in the small intestine.27In the current study the histological severity score indicated that intestinal damage was most severe 72 h after administration of the first injection of MTX, and the pattern of food intake and body weight reflected these changes as reported previously.13,28By 120 h repair had commenced, with mucosal sucrase activity rising, partic-ularly in the ileum. The effects of MTX can be partly ameliorated by factors such as germinated barley,29vitamin A 30and milk derived growth factors.13In the latter two studies mucosal sucrase activity was shown to be a sensitive marker of both the severity of damage and also the extent of its prevention or restoration. In humans with coeliac disease, a condition that exhibits villus damage 31it has been reported that disaccharidase activities, particularly sucrase and maltase were significant positive predictive markers (86–90%) of moderate to severe villus atrophy and an increase in duodenal sucrase activity correlated with recovery of the mucosa on the basis of histological findings. These results therefore support applicability of the SBT to monitor the response of coeliac patients to a gluten-free diet.Reduced brush border enzyme activity may also occur due to inflammation or infection without showing a significant relationship to villus damage.32For example it has been reported that reduced disaccharidase activity can occur when rotavirus-infected intestine exhibits few histopathologic changes.33This is probably due to cytoskeleton alterations which in turn affect translation of the sucrase enzyme to the brush border.34We propose that a low SBT value is indicative of a net reduction of the absorptive capacity of the intestine and a functional reflection of diseased or damaged mucosa.This can only be assessed using an integrated test such as the SBT,whether this change is due to inflammation, infection and/or other mucosal damage. For damage and subsequent repair to the small intestine the time course parallels the expired 13CO 2and when expressed as AUC from 0–90 min, it shows a significant correlation with biochemically-determined sucrase activity. Since the SBT is an integrated measure of net sucrase activity throughout the small intes-tine it is therefore a more practical marker of both damage, and the resultant absorptive capacity, than mucosal sucrase activity and or histological data.In order to evaluate the SBT for its ability to reflect the reduction in sucrase activity throughout the small intestine we chose to assess the effect of an oral dose of folinate administered in the drinking water at the time of MTX administration. Food intake was markedly decreased by MTX treatment compared to normal controls.However, food intake in MTX-treated rats receiving folinate did not differ significantly from controls. Similarly mucosal sucrase activity showed no significant change in the folinate treated group at either 72 h or 120 h post MTX. The SBT results performed on the same animals at 52 h and 100 h respectively were entirely consistent with the biochemically-determined mucosal sucrase activity data and histological analysis of villus integrity.Figure 6. Jejunal MPO activity (U MPO/mg tissue) of rats receiving oral folinate and injected with MTX (n = 4, mean ±SEM; control n = 2). White bar: control;striped bar: MTX + folinate, black bar: MTX no folinate. **,***significant-ly different to MTX no folinate at 72 h (p < 0.01, < 0.001).Figure 4. Jejunal sucrase activity (nmol glucose/min/cm) of rats receiving oral folinate and injected with MTX (n = 4, mean ±SEM; control n = 2).White bar: control; grey bar: MTX + folinate, black bar: MTX no folinate.*control significantly different to MTX no folinate group (p < 0.05); ^ three values above detection level of assay; #two values above detection level ofassay.Figure 5. Area under the curve (AUC) of 13CO 2production of rats receiving oral folinate and injected with MTX (n=4, mean ±SEM). White bar: Pre MTX; striped bar: MTX + folinate, black bar: MTX no folinate. *significant-ly different to preMTX levels (p < 0.05).In order to design adjunctive therapies for mucositis it is important to identify the stages of this condition with particular emphasis on the duration of damage, its extent and the time for the small intestine to return to normal, both histologically and functionally. Previous animal studies using invasive techniques have suggested that IGF-I, for example, may be beneficial when given post-therapy, but may be detrimental if given during therapy.28We have shown that the SBT allows these stages to be monitored non-invasively which in turn will provide rational provision of preventive, ameliorative and reparative agents to be administered at the appropriate time to achieve optimal efficacy. This applies equally to studies involving animal models and humans.In conclusion, this study has demonstrated that the stages of chemotherapy-induced damage, its severity and subsequent intestinal recovery can be effectively and easily monitored by the SBT. The SBT further provides a rapid and non-invasive means of screening candidate anti-cancer drugs for adverse effects on the intestine, in addition to investigating the potential efficacy of newly-developed anti-mucositis agents.References1.Pico JL, Avila-Garavito A, Naccache P. 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