点击显示 收起
1 From the Gastroenterology Unit, Women’s and Children’s Hospital (CDT, GSH, and RNB), the Cooperative Research Centre for Tissue Growth and Repair and the Child Health Research Institute (GSH), the Department of Physiology, University of Adelaide (GSH and RNB), and the Division of Clinical Biochemistry, Institute of Medical and Veterinary Science (PC, JCP, and AMR), Adelaide, Australia.
2 Supported by financial assistance from the Child Health Research Institute and the Gastroenterology Unit, Women’s and Children’s Hospital and by the Institute of Medical and Veterinary Science, which provided infrastructure resources. 3 Address reprint requests to CD Tran, Gastroenterology Unit, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, 5006 SA, Australia. E-mail: tranc{at}mail.wch.sa.gov.au.
ABSTRACT
Background: Oral administration of zinc or bovine whey-derived growth factor extract (WGFE) is known to reduce intestinal permeability and ameliorate methotrexate (MTX)-induced mucositis, respectively.
Objective: We examined the effects of zinc, WGFE, and zinc plus WGFE on gut damage in MTX-treated rats.
Design: Rats (n = 16/group) were fed zinc (1000 mg/kg diet), WGFE (32 mg/kg diet), zinc plus WGFE, or control (10 mg Zn/kg diet) diets for 7 d and then injected subcutaneously with MTX (2.5 mg/kg) for 3 d to induce gut damage. Gut histology and intestinal permeability were assessed.
Results: The Zn+WGFE diet was associated with both reduced gut damage on day 5 and enhanced recovery on day 7. The WGFE diet ameliorated gut damage, whereas the Zn and Zn+WGFE diets enhanced repair. Gut metallothionein and tissue zinc concentrations were significantly (P < 0.01) higher with Zn and Zn+WGFE on days 5 and 7 than without zinc supplementation. The Zn and Zn+WGFE diets significantly (P < 0.05) decreased gut permeability on days 3–4 compared with the control diet. Intestinal permeability was significantly (P < 0.05) increased on days 5–6. On days 6–7, only the WGFE diet improved gut permeability (by 80%) compared with the control diet.
Conclusions: Dietary administration of WGFE and a pharmacologic dose of zinc reduced intestinal damage and enhanced recovery, respectively. WGFE also improved gut permeability after MTX-induced bowel damage. In combination, zinc and WGFE hastened repair of gut damage, which may have clinical application in chemotherapy-induced mucositis.
Key Words: Zinc • whey growth factor extract • intestinal permeability • small-intestine damage
INTRODUCTION
Chemotherapy has been shown to alter mucosal morphology and gut barrier function. Keefe et al (1) and Sundstrom et al (2) showed that chemotherapy used for the treatment of various cancers is associated with increased intestinal permeability to sugar. Intestinal diseases such as inflammatory bowel disease and toxigenic diarrhea are associated with intestinal dysfunction that is linked to enhanced permeability, which indicates that the integrity of the intestinal barrier is altered (3). Zinc supplementation has been shown to reduce intestinal permeability (4, 5) and improve diarrhea in children living in developing countries (6, 7). However, little is known about the influence of zinc supplementation on the modulation of gut integrity and intestinal permeability in chemotherapy-induced intestinal mucositis.
There is now evidence suggesting that growth factors can modify the severity of gut mucositis. Recent studies suggest a protective effect of exogenously administered epidermal growth factor in the prevention of acute changes associated with experimental radiation enteritis (8). The administration of transforming growth factor ß (TGF-ß) has been shown to reduce the severity of oral mucositis after 5-fluorouracil treatment in hamsters (9). Fibroblast growth factors (10) and insulin-like growth factors (IGF; 11), which have been shown to affect gut growth and repair, may also influence the severity of gut mucositis. Treatment with glucagon-like peptide-2 has led to a marked reduction in lesion size in a rodent model of inflammatory bowel disease (12), and keratinocyte growth factor-2 has been postulated to promote healing and inhibit intestinal inflammation by stimulating epithelial restitution and prostaglandin production (13).
Milk is a rich natural source of growth factors that may enhance growth and repair of the gut in newborns (14–16). Human milk is rich in epidermal growth factor, whereas bovine milk contains high concentrations of IGF (15, 16). Milk growth factors have now been extracted and concentrated by cation-exchange chromatography from bovine cheese whey (whey-derived growth factor extract; WGFE) obtained as a by-product of cheese making (17, 18). Howarth et al (19) showed that oral administration of this growth factor extract ameliorated chemotherapy-induced mucositis in rats. Porter et al (20) also showed reduced colonic inflammation in experimental colitis after WGFE administration. The role of growth factors in intestinal growth and function and the interaction between growth factors and specific micronutrients (such as zinc) in improving growth, adaptation, repair, and intestinal permeability of the damaged mucosa are unclear. In the present study, we investigated the effects of pharmacologic dietary supplementation with zinc, WGFE, and a combination of zinc and WGFE (Zn+WGFE) on methotrexate (MTX)-induced gut damage in the rat.
MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats weighing 140 ± 8 g were housed individually in metabolic cages in the animal care facility of the Women’s and Children’s Hospital, which were maintained at 25 °C with a 12-h light-dark cycle throughout the experimental protocol. The protocol followed the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and was approved by the Animal Care and Ethics Committee of the Women’s and Children’s Hospital.
Whey-derived growth factor extract
WGFE was provided by the Cooperative Research Centre for Tissue Growth and Repair and was prepared as described by Francis et al (17). Briefly, fresh cheddar whey was pasteurized (72 °C for 15 s) before microfiltration through a 0.2-µm pore membrane to remove fat. Purification of the growth factor was achieved by Sepharose Fast Flow-S cation exchange chromatography (Pharmacia Biotech, North Ryde, Australia), which takes advantage of the basic isoelectric point of many growth factors, compared with the isoelectric point of the major whey proteins, which is predominantly acidic. The growth factor activity was eluted from the cation-exchange column with 400 mmol NaCl/L and 10 mmol NaOH/L before diafiltration against deionized water, and that activity was followed by concentration. The desalted concentrates were then filtered through a 1-µm glass filter, freeze-dried, and stored at 4 °C. The final WGFE represented an 100-fold concentration of the growth factor content of cheese whey, as determined by cell growth stimulation in cultured Balb/3T3 cells, L6 myoblasts, and human skin fibroblast (17, 18, 21). The WGFE contained 99.2 g protein/100 g WGFE, with lactoperoxidase and immunoglobulins as the most abundant proteins (15).
Zinc and WGFE supplementation and MTX-treated rats
Rats (n = 64) were fed either a purified casein–based control diet (n = 32) or a diet supplemented with 1000 mg Zn/kg diet (Zn diet; n = 32) for 7 d, after which each group was further divided into 2 subgroups of 16 rats. Sixteen rats then continued with the control diet, and the other 16 rats consumed the diet supplemented with WGFE (31.2 g/kg diet; WGFE diet). Similarly, 16 rats of the Zn diet group continued with the same diet, and the other 16 consumed a combination diet containing zinc and WGFE [1000 mg Zn/kg diet and 31.2 g WGFE/kg diet (Zn+WGFE diet)]. The purified casein–based control diet contained 514 g cornflour starch/kg, 180 g casein/kg, 152 g sucrose/kg, 50 g wheat bran/kg, 50 g peanut oil/kg, 2.5 g D,L-methionine/kg, 1 g choline chloride/kg, 4.4 g cod liver oil/kg, and 0.225 g glucose/kg. The mineral profile consisted of 17.155 g KH2PO4/kg diet, 14.645 g CaCO3/kg diet, 12.530 g NaCl/kg diet, 4.99 g MgSO4 • 7H2O/kg diet, 0.296 g FeC6H5O7 • 5H2O/kg diet, 0.170 g CaPO4/kg diet, 0.080 g MnSO4 • 4H2O/kg diet, 0.123 g CuSO4/kg diet, 0.00025 g KI/kg diet, 0.000125 g (NH4)6Mo7 • O24 • 4H2O/kg diet, and 0.00005 g Na2SeO3/kg diet. The vitamin profile consisted of 70 mg thiamin HCl/kg diet, 30 mg riboflavin/kg diet, 50 mg niacin (nicotinic acid)/kg diet, 150 mg pantothenic acid/kg diet, 15 mg pyridoxal HCl/kg diet, 0.02 mg hydroxycobalamin/kg diet, 400 mg inositol/kg diet, 50 mg -aminobenzoic acid/kg diet, 10 mg folic acid/kg diet, and 0.4 mg biotin/kg diet. Zinc was supplemented in the form of zinc chloride. The protein concentration of the control diet was isonitrogenous with that of the WGFE diet.
Animals were injected subcutaneously with 2.5 mg MTX/kg on 3 consecutive days (days 1–3). Rats were maintained on their respective diets until days 5 and 7 after the initial MTX injection, when they were killed (see tissue collection, below). Previous studies (19, 22) showed that MTX-treated rats exhibited maximal intestinal damage on day 5 and that, by day 7, they were exhibiting recovery. These time points were selected as the critical points to assess the potential effect of previous supplementation with zinc, WGFE, and zinc plus WGFE on MTX-induced gut damage. This allowed us to establish whether gut damage was lessened (day 5) or whether there was an increased rate of recovery (day 7). In addition, Howarth et al (19) showed that body weight and food and water intakes were reduced between days 3 and 5 in a MTX time-course study. To compensate for the reduced food intake of rats given MTX between days 3 and 5, ad libitum food intake was allowed, and the rats consumed < 10% of their usual food intake. Any shortfall in food was compensated for by oral gavage of the respective diets to make up for the shortfall. Accordingly, all rats underwent gavage with a 3-mL solution of the corresponding diet from day 3 to day 5 to achieve the same mean daily total levels of intake as those consumed before MTX treatment. Orogastric gavage was performed without anesthesia by passing a polyethylene tube (1.2-mm outer diameter, 0.8-mm inner diameter) through the mouth into the stomach.
Tissue collection
At each time point (days 5 and 7), animals were weighed and asphyxiated by carbon dioxide overdose; blood was withdrawn by cardiac puncture before the rat was euthanized by cervical dislocation. The liver and gut were excised, and the mesentery was removed. The gut was separated into stomach, small intestine, cecum, and colon. The small intestine was further divided into the duodenum, in a section from the gastroduodenal junction to the ligament of Treitz, and the remainder was divided into 3 segments of equal length comprising the jejunum, jejunum-ileum, and ileum. The large intestine was divided equally into the proximal colon and the distal colon. The stomach and cecum were cut open, and their contents were removed by flushing with normal saline; the tissues were then blotted dry and were stored at -70 °C for future measurement of zinc and metallothionein levels. The contents of the small and large intestinal segments were flushed thoroughly with saline, and the first 4–6 cm of each segment was cut and stored as above. The contents of the small intestinal segments were flushed thoroughly with saline, and a 2-cm segment from each region of the small intestine was fixed in methacarn fixative for 2 h before being embedded in paraffin-wax for histologic and cell proliferation assessments.
Zinc and metallothionein analysis
Gut wall segments were diluted 1:5 with 10 mmol Tris buffer/L at pH 8.2 and were homogenized with the use of an Ultra Turrax homogenizer (Janke & Kunkel, Staufen, Germany). A portion of the sample was then boiled and centrifuged at 14000 x g for 3 min at room temperature. The supernatant was analyzed for metallothionein with the use of a 109Cd/hemoglobin affinity assay (23). The remainder of the homogenate was weighed, dried overnight at 80 °C, and then digested with concentrated nitric acid (Aristar; BDH Laboratory Supplies, Poole, United Kingdom) for another 24 h. Zinc in plasma samples and dried gut homogenates subjected to nitric acid digestion were analyzed by atomic absorption spectrophotometry using a Perkin-Elmer 3030 spectrophotometer (Perkin-Elmer Pty Ltd, Uberlingen, Germany).
Assessment of severity scores for intestinal damage
For histologic analysis, paraffin wax–embedded intestinal specimens were sectioned transversely at 4 µm, stained with hematoxylin and eosin, and examined by light microscopy. The semiquantitative histologic assessment for intestinal damage was previously described by Howarth et al (19). Briefly, a total score for each region of the intestine was derived from the sum of scores for 11 histologic criteria. These criteria were villus fusion and stunting (atrophy), disruption of brush border and surface enterocytes, reduction in the numbers of goblet cells, reduction in numbers of mitotic figures, crypt loss and architectural disruption, disruption or distortion of crypt cells, crypt abscess formation, infiltration of polymorphonuclear cells and lymphocytes, dilatation of lymphatics and capillaries, and thickening and edema of the submucosal and muscularis externa layers. One person evaluated the histologic assessments in a blinded manner. Each histologic variable was scored from 0 (normal) to 3 (maximal damage) to give a maximum possible score of 33 for each intestinal region.
Assessment of intestinal permeability with the use of 51Cr-EDTA
Intestinal permeability was assessed by oral administration of 51Cr-EDTA (Amrad Biotech, Victoria, Australia) as described by Davies et al (24). Unanesthetized control animals and rats fed either the Zn, WGFE, or Zn+WGFE diet underwent gavage of 0.5 mL of 10 µCi 51Cr-EDTA/mL that was performed by passing a polyethylene tube (1.2-mm outer diameter, 0.8-mm inner diameter) through the mouth into the stomach. Twenty-four-hour urine samples were collected from the metabolic caged rats after administration of the radiolabeled probe on day 0 (after 7 d of consumption of either a control or zinc-supplemented diet) and on days 3, 5, and 6 after the initial MTX injection. Urine volume was measured and 25 µL of thimerosal (20 mg/mL; Merck Pty Ltd, Victoria, Australia) was added to inhibit bacteria growth. The radioactivity in urine was counted with the use of a gamma counter (Multigamma 1261; LKB Wallac, Turku, Finland). Two standards of a 10 µCi 51Cr-EDTA/mL solution were also counted with each set of urine samples. Relative permeability was determined by calculating the activity present in each urine sample as a percentage of the administered dose after correcting for background radioactivity.
Cell proliferation assessment with the use of bromodeoxyuridine immunohistochemistry
All rats were injected intraperitoneally with 50 mg bromodeoxyuridine/kg (BrdU; Sigma Chemical Co, Castle Hill, Australia) before sacrifice. Incorporation of the thymidine analogue, BrdU, into the DNA occurs during the S-phase of the cell cycle and provides a specific indication of epithelial cell proliferation (25). The BrdU technique was conducted on a single segment of the jejunum-ileum, which is the region maximally affected by MTX treatment. The immunohistochemical staining technique for BrdU detection was performed on methacarn-fixed, paraffin wax–embedded, 3-µm sections of the jejunum-ileum. The tissue sections were incubated in 1% hydrogen peroxide for 30 min to quench any endogenous peroxidase activity and then incubated for 8 min in 1 mole HCl to denature double-stranded DNA. Sections were then blocked with 10% normal rabbit serum diluted in tris-buffered saline for 30 min, rinsed in tris-buffered saline for 10 min, and incubated with mouse anti-BrdU (Dako, Carpenteria, CA) at a dilution of 1:50 (in 1% normal rabbit serum in tris-buffered saline) for 90 min. Sections were then washed twice (in 0.01% Tween 20 in tris-buffered saline) for 15 min, and biotinylated rabbit anti-mouse immunoglobulin G (Dako) was applied at a dilution of 1:400 in tris-buffered saline for 60 min. BrdU-stained jejunum-ileum sections were viewed under a light microscope with the use of a 20x objective. BrdU-labeling indexes for each sample of the jejunum-ileum were determined by counting all of the positively stained epithelial cells in 12–15 well-orientated full-length crypts and then expressing the number as a percentage of the total number of epithelial cells in the 12–15 crypts.
Statistical analysis
For the semiquantitative histologic scoring of intestinal damage, data are expressed as geometric means with ranges, and, for all other measurements, data are expressed as means (± SEMs). The semiquantitative histologic scores and intestinal permeability data were log transformed to normalize the data and reduce the variance. All data were then analyzed with the use of a two-way analysis of variance to determine whether there was a significant difference across dietary treatment groups and across regional sites of the gut within each dietary group. Two-way analysis of variance was also performed to compare plasma zinc levels in treatment groups between days 5 and 7. When differences were detected, group means were compared with the use of Bonferroni’s post hoc test. Marginal means (means of rows and columns within the table) were included when interaction was not significant. Differences were considered to be significant at P < 0.05. All statistical analyses were performed with SIGMASTAT statistical software, version 2.03 (SPSS Inc, Chicago).
RESULTS
Severity scores
Food intake during the experimental protocol did not differ significantly in the Zn (17 ± 2 g/d), WGFE (18 ± 1 g/d), and Zn+WGFE (17 ± 3 g/d) dietary interventions from that in the control dietary intervention (18 ± 2 g/d), except during the period of severe gut damage. Semiquantitative assessment of the histologic severity of gut damage from MTX treatment on day 5 showed that rats consuming the WGFE or Zn+WGFE diet had significantly (P < 0.05) lower severity scores in the jejunum (by 30% and 32%, respectively) and ileum (by 27% and 48%, respectively) than did those consuming the control diet (Table 1).
View this table:
TABLE 1 . Semiquantitative histologic assessment of methotrexate (MTX)-induced intestinal damage in different regions of the small intestine 5 d after the first MTX injection in 8 rats1
On day 7, the severity scores in animals fed the Zn+WGFE diet were 58%, 45%, 47%, and 45% lower in the duodenum, jejunum, jejunum-ileum, and ileum, respectively, than were the scores in animals fed the control diet (P < 0.05; Table 2). The severity of damage in the ileum of animals fed the Zn diet alone was significantly (P < 0.05) lower (53%) than that in the animals fed the control diet.
View this table:
TABLE 2 . Semiquantitative histologic assessment of methotrexate (MTX)-induced intestinal damage in different regions of the small intestine 7 d after the first MTX injection in 8 rats1
Gut zinc
Animals fed the Zn or Zn+WGFE diet had gut zinc concentrations on day 5 that were, on average, 4- and 3-fold, respectively, higher (P < 0.01) than the zinc concentrations in animals fed the control diet (Table 3). On day 7, gut zinc concentrations were still significantly (P < 0.05) higher in most regions of the gut, except the proximal small intestine, in rats fed the Zn and Zn+WGFE diets than were the concentrations in rats fed the control and WGFE diets (Table 4). On average, zinc concentrations after treatment with the Zn and Zn+WGFE diets were 3-fold higher than those after treatment with the control and WGFE diets (Table 4).
View this table:
TABLE 3 . Zinc concentrations in segments of the gut wall with methotrexate (MTX)-induced damage 5 d after the first MTX injection in rats fed various diets for 7 d previously and throughout the MTX treatment1
View this table:
TABLE 4 . Zinc concentrations in segments of the gut wall with methotrexate (MTX)-induced damage 7 d after the first MTX injection in rats fed various diets for 7 d previously and throughout the MTX treatment1
Rats fed the Zn and Zn+WGFE diets had significantly (P < 0.05) higher plasma zinc concentrations (Zn diet: day 5, 61.2 ± 5.6 µmol/L; day 7, 39.3 ± 6.1 µmol/L and Zn+WGFE diet: day 5, 60.7 ± 6.1 µmol/L; day 7, 38.5 ± 3.1 µmol/L) than did rats fed the control diet (day 5, 17.8 ± 1.1 µmol/L; day 7, 12.9 ± 0.6 µmol/L) and the WGFE diet (day 5, 17.9 ± 1.6 µmol/L; day 7, 14.0 ± 0.8 µmol/L).
Gut metallothionein
MTX-treated rats fed the Zn or Zn+WGFE diet had significantly (P < 0.01) higher metallothionein concentrations along the gut on day 5 than did rats fed the other diets (Table 5). Gut metallothionein concentrations in the duodenum and colon on day 7 were significantly (P < 0.001) higher in rats fed the Zn or Zn+WGFE diet than in those fed the control diet (Table 6).
View this table:
TABLE 5 . Gut metallothionein concentrations in segments of the gut wall with methotrexate (MTX)-induced damage 5 d after the first MTX injection in rats fed various diets for 7 d previously and throughout the MTX treatment1
View this table:
TABLE 6 . Gut metallothionein concentrations in segments of the gut wall with methotrexate (MTX)-induced damage 7 d after the first MTX injection in rats fed various diets for 7 d previously and throughout the MTX treatment1
Assessment of intestinal permeability with the use of 51Cr-EDTA
Intestinal permeability was assessed by measuring 24-h urinary excretion of labeled 51Cr-EDTA in MTX-treated rats fed any of the study diets (Table 7). Intestinal permeability was measured on days 0–1 (before MTX treatment) and on days 3–4, days 5–6, and days 6–7 after the initial MTX injection. On days 0–1, there was no difference in intestinal permeability in control or Zn diet-fed rats. However, after the 3 MTX injections (days 3–4), rats fed either the Zn or Zn+WGFE diet had significantly (P < 0.05) less intestinal permeability (63% and 65%, respectively) than did MTX-treated rats fed the control diet. By days 5–6, much of the intestinal epithelium was excoriated, as shown by the marked increase in permeability, and there was no significant difference in permeability between treatments. By days 6–7, intestinal permeability was still increased in the controls. On days 6–7, intestinal permeability had almost returned to day 0 levels in the WGFE diet group and was 80% lower (P = 0.006) than in the control diet group.
View this table:
TABLE 7 . Percentage of control of 24-h urinary excretion of labeled 51Cr-EDTA before methotrexate (MTX)-induced intestinal damage and 0–1, 3–4, 5–6, and 6–7 d after the first MTX injection in rats fed various diets for 7 d previously and throughout the MTX treatment1
BrdU labeling, crypt depth, and villus height
BrdU labeling was performed on the jejunum-ileum segment on days 5 and 7 after the initial MTX injection. Epithelial proliferation in the intestine of rats on day 5 or day 7, respectively, after consumption of the Zn (
DISCUSSION
The present study showed that WGFE supplementation with or without zinc had beneficial effects on the gut on day 5 after MTX-induced damage. Our findings showed that WGFE prevented gut damage, whereas the addition of zinc gave no further benefit, which is consistent with the findings of Howarth et al (19) that the administration of WGFE improved villus surface indexes in the jejunum and ileum by 52% and 56%, respectively, and in the jejunal crypt area index by 64%. They postulated that WGFE may have caused lengthening of intact crypts or increased regeneration of new crypts. Presumably, one or more of the growth factors in WGFE contributed to its protective role; these include IGF, platelet-derived growth factor, and fibroblast growth factors, all which have been implicated in the wound healing process (26). In addition, IGF-I has been shown to enhance epithelial proliferation in rat intestine (27, 28).
In contrast, during recovery from MTX-induced gut damage (day 7), Zn+WGFE supplementation appeared more effective in promoting the recovery of gut integrity, whereas zinc alone or WGFE alone did not enhance repair except in the ileum. It is not clear how zinc and growth factor extracts act in combination to improve gut integrity. It may be that nutrients and growth factors interact to regulate the expression or function of mucosal nutrient transport, enhance the antioxidant capacity of gut mucosa, or increase local growth factor production or action (or both) to facilitate gut growth and function (26). Ninh et al (29) reported that zinc supplementation was associated with increased plasma IGF-I concentrations in malnourished Vietnamese children and suggested that the growth-stimulatory effects of zinc could be mediated through changes in circulating IGF-I. Moreover, TGF- has been shown to act as a mitogen and induce expression of 2 zinc finger proteins [Zif268 (zinc finger protein 268) and Nup475 (nuclear protein 475)] in rat intestinal epithelial cells in culture and in the rat intestine (30). Thus, growth factors can interact with genes coding for zinc finger proteins that are involved in regulating intestinal epithelial growth or repair or both. Although, TGF- is not found in WGFE, its epidermal growth factor family peptide member, betacellulin, has been isolated (31). TGF- and betacellulin have a similar molecular weight, share amino acid sequence homology, compete for binding to a common receptor, and induce similar biological responses (32). The improvement in mucosal integrity may therefore have been mediated by the interaction between Zn and betacellulin.
In the present study, WGFE had a significant effect on enhancing functional recovery after MTX, as indicated by improvement in intestinal permeability; the greatest reduction (80%) showed that WGFE was more effective than zinc alone in this regard. Colostrum, which also contains an array of growth factors similar to WGFE, has been shown to reduce gut permeability caused by nonsteroidal antiinflammatory drugs in healthy subjects (33). In addition, colostral preparations given orally reduced gastric injury by 60%, which is similar to the reduction found when TGF-ß was administered to rats (34). In the current study, it was noted that zinc had a more rapid onset of action than did WGFE, as reflected by an improvement in permeability after only 3 d of administration, which was simultaneous with the administration of MTX. However, the improvement on days 6–7 that was attributed to WGFE was apparent after 6 d of WGFE supplementation, whereas on days 6–7, zinc had been administered for 14 d. This effect of WGFE does not correlate with the tissue concentrations of zinc or with the induction of metallothionein, but increased absorption of these growth factors at the tissue level may have provided the accelerated action of WGFE compared with that of zinc in the repair phase.
It is not clear how growth factors affect mucosal barrier integrity, although there is likely to be a local effect on the intestinal mucosa. Growth factors may modify intestinal permeability by binding to cytoskeletal proteins such as zonula occludens, junctional adhesion molecules, and other tight junction-associated proteins, thereby influencing paracellular permeability. Moreover, a generalized effect of growth factors may play a role by inhibiting oxidative stress–responsive transcription factors activated in inflammatory disease states (35) or by increasing cell proliferation in the regenerating mucosa. Growth factors—in particular, epidermal growth factor and TGF-—protect against damage by preventing the disruption of the microtubule cytoskeleton, in which action the activation of protein kinase C signaling and normalization of intracellular Ca2+ appear to be key mechanisms (36).
In the current study, feeding a diet with zinc alone or Zn+WGFE markedly increased plasma zinc concentrations, as well as tissue zinc concentrations, but this did not ameliorate the maximal mucosal damage induced by MTX. This finding is consistent with that of Di Leo et al (37), who reported that zinc administration had little effect on the short-term course of experimental colitis in rats, when assessed histologically. The dietary zinc administered in the present study was at a pharmacologic dose, which is well tolerated in rats and known to induce metallothionein (38). Metallothionein has been shown to sequester free radicals in inflammatory disease states (39). Thus, induction of metallothionein may have further protected the small intestine from MTX-induced inflammation.
The disparity between mucosal damage assessed histologically and changes in intestinal permeability after WGFE supplementation may not be inconsistent. The WGFE-supplemented rats entered the repair phase earlier or had a higher rate of repair (or both), such that, on days 6–7, there was a clear improvement in gut permeability in these animals. In contrast, histologic severity scores on day 7 did not reflect the improvement in intestinal permeability in the WGFE-supplemented animals; only with the addition of zinc was there a significant recovery of gut integrity. This suggests that WGFE requires the presence of zinc to maintain the growth and repair of damaged mucosal cells, whereas WGFE alone is able to improve tight junctions between cells. Supporting this premise is the fact that intestinal permeability primarily reflects the integrity of the epithelium, particularly the tight junctions, and as such is an early phenomenon, preceding histologic and biochemical changes (40). On the other hand, the histologic score is the sum of both the morphologic and architectural disruptions of the mucosa and includes the inflammatory response indicated by the infiltration of polymorphonuclear cells and lymphocytes, which together represent the later phase of gut damage. The histologic score on day 5 thus represented maximal mucosal damage in all MTX-treated rats, irrespective of diet supplementation, and therefore it is perhaps not surprising that a reduction in intestinal permeability was not found with any dietary intervention.
In conclusion, feeding a Zn+WGFE diet significantly improved gut recovery from MTX-induced damage. Zinc was more effective than WGFE in preventing mucosal damage, whereas WGFE was more effective than zinc in improving mucosal recovery. The combination of zinc and WGFE, however, resulted in the greatest improvement. The dietary regimen of WGFE followed by the combination of zinc and WGFE may reduce the severity of the MTX damage and thus result in improved permeability indexes and a more rapid return to normal by the intestinal mucosa. The results of the present study provide evidence that orally administered zinc used in combination with WGFE may be useful in the treatment of intestinal mucositis induced by high-dose chemotherapy.
ACKNOWLEDGMENTS
CDT performed all the experimental work, data collection and entry, analysis of results, and statistical analysis and had a major role in writing the report; RB oversaw the experimental work and preparation of this report; GH was responsible for the animal model and aspects of work related to WGFE; and PC, JP, and AR were responsible for the zinc and metallothionein-related aspects of the project. All of the coauthors had input into the study design and in the writing and editing of the manuscript. None of the authors had a conflict of interest related to this work.
REFERENCES