1

Methotrexate induces intestinal mucositis and alters gut protein metabolism

independently of reduced food intake.

Nabile Boukhettala 1, Jonathan Leblond 1, Sophie Claeyssens 1,2, Magali Faure 3, Florence

Le Pessot 4, Christine Bôle-Feysot 1, Aktham Hassan 1, Christine Mettraux 3, Jacques

Vuichoud 3, Alain Lavoinne 1,2, Denis Breuillé 3, Pierre Déchelotte 1 and Moïse Coëffier 1

1 ADEN – EA4311, Institute for Biomedical Research and European Institute for Peptide

Research (IFRMP23), Rouen University and Rouen University Hospital, Rouen, France.

2 Laboratory of Medical Biochemistry, Rouen University Hospital, France.

3 Nestlé Research Center, Nutrition & Health Department, Lausanne, Switzerland.

4 Laboratory of Anatomo-Pathology, Rouen University Hospital, France.

Correspondence:

Moïse Coëffier

ADEN EA4311, Institute for Biomedical Research, IFRMP23, 22 boulevard Gambetta 76183

Rouen cedex 1, France.

tel: +33 2 35 14 82 40 ; Fax: +33 2 35 14 82 26 ; @: moise.coeffier@univ-rouen.fr.

Short title:

Gut protein metabolism during intestinal mucositis

Financial support:

Nestlé Research Center, Nutrition & Health Department, Lausanne, Switzerland.

Articles in PresS. Am J Physiol Endocrinol Metab (November 11, 2008). doi:10.1152/ajpendo.90459.2008

Copyright © 2008 by the American Physiological Society.

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Conflict of interest

None to declare

Abbreviations:

FSR: fractional synthesis rate

GSH: glutathione

IFNγ: interferon γ

IL-1β: interleukin 1 β

IL-4: interleukin 4

IL-6: interleukin 6

IL-10: interleukin 10

MTX: methotrexate

qPCR: real-time quantitative - polymerase chain reaction

TNFα: tumor necrosis factor α

Key Words:

Chemotherapy, Intestine, Nutrition, protein metabolism, mucin.

3

Abstract:

One of the main secondary toxic side effects of antimitotic agents used to treat cancer patients

is intestinal mucositis. This one is characterized by compromised digestive and absorptive

functions, barrier integrity and immune competence. In the same time, food intake is

decreased and may induce intestinal damages per se. The aim of the study was to characterize

which alterations are specific to methotrexate, independently of the anorexic effect of the

drug. Male Sprague-Dawley rats received subcutaneously saline solution as control group or

2.5 mg/kg of methotrexate during three days (D0-D2). Methotrexate-treated rats were

compared to ad libitum and pair-fed controls. Histological examinations and specific markers

of the immune and non-immune gut barrier function were assessed at D4 or D7. As compared

to ad libitum and pair fed controls, methotrexate induced at D4 villus atrophy associated with

epithelial necrosis. Mucosal protein synthesis rate and mucin contents of methotrexate treated

rats were reduced. In the same time, cathepsin D proteolytic activity was increased compared

with ad libitum and pair fed controls whereas calpain activity was increased when compared

with the only pair fed controls. These intestinal lesions were associated with various

metabolic disturbances such as increased TNF-α level and inflammation score in the jejunum

but also disturbances of amino acid concentrations in duodenum and plasma. At D7, these

alterations were partially or completely normalised. In addition to consequences of a low food

intake, methotrexate further impairs different biological processes leading to a dramatic loss

of gut homeostasis. Targeted nutritional management of chemotherapy receiving patients

should be set up to prevent or limit such alterations.

4

Introduction:

Numbers of anticancer agents are anti-mitotic drugs such as methotrexate (MTX) or 5-

fluorouracil. These drugs act by blocking DNA and RNA synthesis, induce apoptosis and

slow down or stop cell proliferation (20). Since their actions are not specific to tumor tissues,

they also induce deleterious effects on rapidly proliferating cells such as bone marrow and gut

mucosa cells. Hematopoietic, oral and gastrointestinal toxicities are the most frequently

observed and may lead to a reduction in the dose or even a cessation of chemotherapeutic

treatments. The bone marrow suppression may be treated with growth factors or even

compensated by bone marrow transplantation (32). In contrast, there is no efficient standard

treatment to reduce oral and gastrointestinal injuries called intestinal mucositis. The main

clinical features of intestinal mucositis include nausea, bloating, abdominal pain and diarrhea.

These symptoms generally peak on day 3 after chemotherapy initiation but oral symptoms are

still maximum on day 14 with pain and erythema (22). So, intestinal mucositis remains a

major concern during cancer chemotherapy in more than 40% of cancer patients after standard

doses of treatment (21), and in almost 100% of patients treated with high doses (33). In the

gut, mucosal damage and barrier function alterations have been described as consequences of

different processes: apoptosis, hypoproliferation, inflammatory response, altered absorptive

capacity and bacteria proliferation and colonization (6).

A decrease of food intake is also observed during chemotherapeutic treatments. This

one could be due to painful mucosal lesions (34) but the central regulation of food intake

could also contribute to this anorexic effect. During experimental colitis, pro-inflammatory

cytokines have been associated with serotonin release from the hypothalamic paraventricular

nucleus (24, 38), and may induce a reduction of food intake (2). Low food intake is known to

induce rapid and profound wasting of several organs (30, 36), especially in the small intestine,

which is more sensitive to food restriction due to its very high rate of protein turnover (4, 5,

5

9). For instance, food restriction is associated with an increase of intestinal permeability (42),

of inflammatory response (29) and intestinal proteolysis in rat intestine (36) and with a

decrease of glutathione content (3). We recently reported increased intestinal permeability,

mucosal damages and proteolysis alterations in rats treated with MTX-induced enterocolitis.

We observed in the same time that MTX induced a marked decrease of food intake in rats

(26). However, we did not assess in this study whether these alterations were directly due to

MTX treatment or to reduced food intake.

In the present study, we characterized the specific effects of MTX treatment

independently of the food intake effect. For that, we compare the effect of MTX treatment on

mucosal damages, intestinal protein balance (protein synthesis and degradation), mucin and

glutathione (GSH) content to pair-fed control rats.

6

Materials & Methods:

Animals, Housing, Diet & MTX injections

Animal care and experimentation complied with both French regulation and European

Community regulation (Official Journal of the European Community L 358, 18/12/1986) and

MC is authorized by the French government to use animal model (Authorization n° 76-60).

During 1 week, 200-250 g male Sprague-Dawley rats (Charles River, L’Arbresle, France)

were acclimatized at 25°C with a 12 hours light-dark cycle. Animals were individually housed

in metabolic cages four days before study. Rats were given free access to water and standard

chow until day -3. Afterwards, MTX-treated rats had free access to a protein-rich diet.

Control rats were either ad libitum or pair-fed versus MTX rats with the same diet. Rats were

subcutaneously injected during the three first days (D0, D1 and D2) with 2.5 mg/kg MTX

(Teva Pharma, Courbevoie, France) or NaCl solution (0.9%) as control [16]. Rats were

euthanized on D4 or D7 for pair fed and MTX treated rats and at D7 for ad libitum rats. D4

and D7 were chosen to be in the acute (D4) and the recovery (D7) phases of mucositis after

preliminary experiments (data not shown).

Daily Clinical State Monitoring

From D-3 to euthanasia day, body weight and food intake were monitored at 24-hours

intervals.

Euthanasia and Tissue Sampling

Animals were decapitated after carbon dioxide inhalation and blood samples were

collected. Plasma samples were stored at -80°C as previously described (14). Then, the

duodenum and the jejunum were taken and rinsed with ice-cold PBS (140 mM NaCl, 3 mM

KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4). Duodenum was directly frozen in liquid nitrogen

until amino acid assessment. For histological and immunohistochemistry assessments, 2

proximal samples of jejunum (1 cm long each) were fixed in formalin (10%). A piece (1 cm

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long each) was removed for mucin content analysis. Four consecutive pieces (2 cm long each)

were respectively removed and mucosa was scraped for the determination of protein synthesis

rate, of the determination of proteolytic activities, the mRNA expression and the analysis of

cytokine concentrations. Then middle pieces (1 cm long) were removed for assessment of

GSH content. Samples were immediately frozen in liquid nitrogen and stored at -80°C. For

mRNA, TRIZOL® Reagent (Invitrogen, Cergy-Pontoise, France) was previously added to

collecting tubes.

Histological Parameters

For histological and immunohistochemistry assessments, jejunal samples were fixed in

formalin 10% and treated as previously described (26). Briefly, sections were scored by the

same pathologist blinded to the treatment allocation. Epithelial necrosis, inflammatory cells

infiltration and exocytosis were assessed using semiquantitative scores which ranged from 0

(no damage) to 3 (severe damages) for each parameter: villus atrophy, necrosis, inflammation

and exocytosis. Villus height was measured on 10 well-oriented villi from each rodent using

the analysis software Leica QWin (Leica Microsystems, Bensheim, Germany).

Evaluation of Cell Proliferation

The rate of cell proliferation was evaluated by the incorporation of 5-bromo-2-

deoxyuridine (60 mg/kg, BrdU, Sigma-Aldrich, Saint-Quentin-Fallavier, France) injected

intraperitoneally one hour before euthanasia as previously described (26). Briefly,

immunohistochemistry was performed on fixed and embedded in paraffin tissue with BrdU

monoclonal antibody (dilution 0.1, M0744, DakoCytomation, Trappes, France) according to a

standard immunohistochemical method using a streptavidine-biotin/horseradish peroxydase

detection system (5001, DakoCytomation). BrdU detection was then scored from 0 (no

labelling) to 3 (high level of labelling).

Intestinal Cytokine Concentrations and Glutathione content

8

Intestinal cytokines concentrations were evaluated by specific ELISA assay

(Quantikine, R&D Systems, Abington, UK) for IL-1β (interleukin 1β), IL-4 (interleukin 4),

IL-6 (interleukin 6), IL-10 (interleukin 10), IFNγ (interferon γ) and TNFα (tumor necrosis

factor α) according to manufacturer’s instructions and as previously described (26).

Concentrations were expressed as pg/mg tissue.

GSH content was assessed in the jejunum by a standardized spectrophotometric assay

as previously described (1, 26). GSH content was expressed as µmol/g tissue.

Amino Acid Concentrations:

Free amino acid concentrations were determined in plasma and in the duodenum as

previously described (14).

Evaluation of Mucosal Protein and Mucin Contents

The protein content in the jejunal mucosa was assessed using the Bio-Rad dye reagent

kit (Bio-Rad Laboratories, Reinach, Switzerland). For mucin quantification, jejunal samples

were processed, and mucins quantified as previously described (13). The mucin content of

each sample was then correlated with the weight of the initial tissue an expressed as µg

mucins/g tissue.

Evaluation of Mucin Gene Expression by Real-Time Quantitative – Polymerase Chain

Reaction (qPCR) on TaqMan

TaqMan qPCR was carried out in a ABI 5700 Thermocycler (Applied Biosystems,

Rotkreuz, Switzerland) using total RNA extracted from whole-thickness jejunum using the

Tripure Isolation reagent (Roche Diagnostics AG, Rotkreuz, Switzerland), analyzed with an

Agilent 2100 Bioanalyzer and the RNA 6000 Nano LabChip kit (Agilent Biotechnologies,

Waldbronn, Germany) for quality verification, and reverse transcribed. Primers and TaqMan

probes for mucin genes were designed (Supplemental Table S1) and synthesized for mucin 2,

mucin 3 and mucin 4. The Taqman probes presented in 5’ end an additional 6-

9

carboxyfluorescein. The reactions were carried out in triplicate. Data were analyzed using the

GeneAmp 5700 SDS software (Applied Biosystems). Relative gene expression was calculated

after normalization to β2-microglobulin whose primers and TaqMan probe were purchased

from Applied Biosystems.

Protein Metabolism Study

1) Protein Synthesis: In vivo protein synthesis was assessed by the infusion of a large dose of

L-[ring-2H5]-phenylalanine (1500 µmol/kg, 90% MPE, Mass Trace Inc., Woburn, MA) via a

lateral tail vein as previously described (16). Animals were killed 10 min after the tracer

administration. Tissues were immediately excised and the mucosa of the jejunum were

rapidly separated and frozen in liquid nitrogen. Under these conditions, plasma-free

phenylalanine enrichment decreased slightly and linearly as it has been previously verified in

rats killed at various time (Figure 1). The enrichments of [2H5]-phenylalanine was determined

in the mucosal intracellular free amino acid pools and in the tissue proteins, by gas-

chromatography-mass spectrometry (MSD 5975, Agilent Technologies, Palo Alto, CA), using

tert-butyldimethylsilyl derivatives as previously reported (4, 8). Appropriate standard curves

were run simultaneously for determination of the enrichments. Protein synthesis rate was

expressed as fractional synthesis rate (FSR) calculated as follows: FSR (% / day) = [(Et-Eo) /

Ep] x 1 / t x 24 x 100, where Et is the enrichment in tissue protein at time t, in % ; Eo is the

natural abundance of the labelled amino acid in intestinal mucosal protein, in % ; Ep is the

enrichment of intracellular free amino acid pool at time t; "t" is the duration of the tracer

infusion, in hour(s).

2) Proteolysis: Evaluation of proteolytic pathway activities was performed by measuring

fluorogenic substrate degradation, as previously described (25, 26). Briefly, lysosomal

cathepsin D activity was quantified with InnoZyme™ Cathepsin D Immunocapture Activity

Assay Kit Fluorogenic (Calbiochem, San Diego, CA). Evaluation of calpain (1 and 2) and

10

proteasome chymotrypsin-like activity was performed by spectrofluorimetry on microtiter

plate fluorometer Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany) using

fluorogenic proteasome substrate, Suc-LLVY-MCA (PSIII, Calbiochem) in the presence or

absence of specific inhibitors.

Evaluation of gene expressions for proteolytic pathway components was realized by

qPCR on LightCycler™ as previously described (26).

Calculation Methods and Statistical Analysis

Statistical analysis was performed using GraphPad Prism 5.01 (GraphPad software

Inc, San Diego, CA) and consisted in a two-way ANOVA with Bonferroni post-hoc tests

when appropriate. For daily monitored parameters, repeated measures two-way ANOVA was

performed. For all, p<0.05 was considered significant.

11

Results:

Food Intake and Body Weight.

After MTX treatment, food intake decreased until D5 (-60% at D2, -96% at D5, both

p<0.0001 vs D0) and then increased (-30% at D7, p<0.0001 vs D5). No difference in food

intake was observed between pair fed and MTX groups (data not shown).

We compared body weight evolution of ad libitum, pair fed and MTX rats during 11

days from D-3 to D7 (Figure 2). Before MTX or saline treatment, body weight increased in

the three groups at the same rate (5%). After injection of rats with 2.5 mg/kg of MTX or

saline, body weight increased over time in ad libitum rats but progressively decreased from

D2 until D6 (about -15%) in a similar manner in pair-fed and MTX-treated rats. Thus, body

weight was significantly reduced in pair-fed and MTX-treated rats compared with ad libitum

controls.

Histological damages and cellular proliferation in the jejunum.

Intestinal morphometry (Figure 3) were similar in pair fed group at D4 and D7

compared with ad libitum controls. MTX-treated group presented a marked decrease of villus

height at D4 (Figure 3A-B) and an increase of inflammation score (Figure 3C) compared with

ad libitum and pair-fed groups. In MTX-treated rats, necrosis score was 1.7 ± 0.2 at D4 and

0.7 ± 0.3 at D7, whereas any necrosis event was observed in control ad libitum and pair fed

rats. At D7, villus height was restored to that of ad libitum and pair-fed rats and inflammation

score began a decrease.

The cellular proliferation rate did not significantly differ between pair-fed and MTX-

treated rats but was increased at D7 as compared to D4 (Table 1).

Gut protective factors: mucins and glutathione.

The mucin content was markedly impaired by MTX treatment compared with pair fed

group (Table 1). At D4, in MTX-treated rat mucosa, the MUC2 gene expression was

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significantly reduced (p<0.05) while thereafter significantly increased (p<0.01) at D7 (Figure

4). In contrast, the MUC3 and MUC4 gene expressions were significantly increased (p<0.05)

both at D4 and D7 (Figure 4).

Glutathione content was similar in pair fed and MTX-treated rats and increased at D7

in both groups (Table 1).

Inflammatory response.

TNFα concentration in jejunal mucosa was higher in MTX-treated rats than in ad-

libitum and pair-fed rats (p<0.05, Figure 5A). In contrast, IL-1β concentration was increased

in pair fed and MTX-treated rats compared with ad libitum controls but no difference was

observed between pair fed control rats and MTX-treated rats (Figure 5B). IL-4, IL-10 and

IFNγ remained undetectable in the majority of jejunal samples (data not shown).

Protein Metabolism

Plasma and duodenal amino acid concentrations were respectively displayed in Tables

2 and 3. The food restriction imposed to pair fed rats was associated with modifications of the

concentrations of various amino acids at day 4 and 7. More importantly, at D4, plasma

concentrations of citrulline, isoleucine and threonine were lower in MTX-treated rats than in

control ad libitum and pair-fed rats. At D7, only citrulline concentration remained low. By

contrast, plasma concentrations of alanine and proline were increased in MTX-treated rats at

D7 as compared to control ad libitum and pair-fed rats.

In duodenum, alanine, aspartate, isoleucine, leucine, lysine, ornithine, proline,

threonine and valine concentrations were similarly reduced at D4 in control pair fed and

MTX-treated rats (table 3). Citrulline concentration decreased both in control pair fed and

MTX-treated rats compared with ad libitum rats but more markedly in MTX-treated rats. In

contrast, glutamine concentrations were increased both at D4 and D7 in MTX-treated rats

compared with control ad libitum and pair fed rats.

13

Protein content of jejunum was significantly reduced both in pair fed and MTX groups

compared with ad libitum group at D4 and D7 (Figure 6A). The decrease was more marked in

MTX treated rats at D4 compared with pair fed rats but the difference did not reach

significance. In addition, protein content increased between D4 and D7 in MTX treated rats.

Thus, we evaluated protein synthesis and protein degradation.

Protein fractional synthesis rate (FSR) was significantly decreased in MTX-treated rats

compared with ad libitum and pair-fed rats at D4 (p<0.01, Figure 6B) and was restored at D7.

There was no difference between ad libitum and pair fed controls.

At D4, cathepsin D activity, a component of the lysosomal proteolytic pathway, was

significantly increased both in pair-fed and MTX-treated rats at D4 and D7 compared with ad

libitum controls (p<0.05) (Figure 7A). Moreover, cathepsin D activity was significantly

increased in MTX-treated rats compared with pair-fed rats both at D4 and D7 (p<0.05, Figure

7A) even if cathepsin D activity decreased significantly between D4 and D7 in MTX-treated

rats. Cathepsin D mRNA level was also increased in MTX-treated rats compared with pair

fed rats (supplemental Table S2).

Regarding the Ca2+-activated pathway, calpain activities were significantly increased

in MTX-treated rats at D4 compared with pair-fed rats but not compared with ad libitum rats

and then decreased at D7 (Figure 7B). Calpain 1 mRNA was affected both by treatment and

day, being higher in MTX-treated rats compared with control pair-fed rats and increasing

between D4 and D7 in both groups (supplemental Table S2). Calpain 2 mRNA level was

significantly increased by MTX treatment but without any day effect (supplemental Table

S2).

At D4 and D7, the proteasome chymotrypsin-like activitiy were significantly

decreased in pair-fed and MTX-treated rats compared with ad libitum rats (p<0.001, Figure

7C). However, this activity did not differ significantly between MTX-treated and pair-fed rats

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at D4 (Figure 7C). Between D4 and D7, proteasome activity increased significantly in the

pair-fed group but not in MTX-treated rats. Consequently proteasome activity was lower in

MTX-treated rats than in pair-fed rats at D7. Ubiquitin mRNA expression, a key component

of proteasomal degradation, increased after MTX treatment (supplemental Table S2).

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Discussion:

Mucositis, a severe side effect of chemotherapy, may cause reduction or interruption

of cancer treatment. Thus, a better understanding of its pathophysiological mechanisms is

required to develop adapted therapeutic strategies. We previously reported alterations of the

proteolytic pathways during chemotherapy-induced enterocolitis in rats (26), but whether

these alterations were due to chemotherapeutic drugs or to the decrease of food intake which

could be related to gastrointestinal pains or to direct or indirect central effects of drugs

remains unknown. Indeed, in this precedent study, the control group was fed ad libitum.

Therefore we investigated in the present study the specific effects of MTX on the intestine

independently of the reduction of food intake using ad libitum and pair-fed rats as controls.

In the present study, the marked reduction of food intake resulted in a rapid (-10% at

D4) and profound (-20% at D6) decrease of body weight in rats as previously described (17).

Surprisingly, we did not observe a further decrease of body weight in MTX-treated rats

compared with pair-fed rats, indicating that body mass loss observed after MTX treatment

would originate from food intake reduction per se.

In contrast, MTX-treated rats presented major histological mucosa damages in the

jejunum as compared to ad libitum and pair-fed rats. In particular, MTX-treated rats showed

necrosis processes that were not present in pair-fed rats. Moreover a strong reduction of villus

height was observed in MTX-treated rats. In the same time, citrulline concentration was

dramatically low in duodenum and plasma of MTX-treated rats. This was especially the case

on D4 since citrulline values of MTX treated animals represent only 24 and 18% of control

pair-fed values, respectively in duodenum and plasma. Interestingly, intestinal mucosa

atrophy was correlated with reduced plasma citrulline concentration (Figure 8). In MTX-

treated rats, the low concentration of this non protein amino acid, synthesized by enterocytes

could reflect reduction of the enterocyte mass as previously described in different diseases

16

affecting the gut (11, 12) and could suggest a reduction of absorptive function. Our results

demonstrate that MTX treatment induced a mucosal atrophy correlated with the citrulline

decrease, independently of the anorexic effect of the drug.

To explain these histological differences, we compared protein metabolism, as well as

the mucosal status in two key players of host defense mechanisms, namely mucins and GSH

in the jejunum of MTX-treated vs pair-fed rats.

Mucosal damages could be related to alterations of protein metabolism and we

previously reported that MTX treatment was associated with enhanced lysosomal proteolysis

and decreased proteasome activity when compared with ad libitum rats (26). However mRNA

expressions of proteolytic pathway component such as ubiquitin, and C2, C8, C9 subunits

(proteasome) or cathepsin D (lysosome) and m-calpain (Ca2+-activated proteolytic pathway)

were significantly increased by 48 hours of starvation in the whole jejunum (19, 36), but not

in the jejunal mucosa (35). In the present study, we show that during the acute phase of

enterocolitis (D4), cathepsin D-mediated proteolysis, which was considered as a marker of

lysosomal activity, was enhanced specifically by MTX treatment as previously described

(26). We also observed that calpain activities were increased but only compared with pair fed

rats. These parameters were completely or partially restored at D7. In contrast, proteasome

activity remained low at D4 and D7 in pair-fed and MTX-treated rats. In addition, calpain

activities were correlated with the mRNA level of calpain 2 (r=0.3, p<0.05, data not shown),

suggesting that calpain activities was mainly related to calpain 2. Taken together, these results

suggest that the main proteolytic pathway that was activated in the jejunum after MTX

treatment was the cathepsin D system whatever the rats used as control, namely pair-fed or ad

libitum animals in the present study, or ad libitum animals in our previous work (26). In

inflammatory bowel diseases, cathepsin D is enhanced in macrophages of gut mucosa (18). In

animal model of inflammatory bowel diseases, cathepsin D inhibition by drugs limits

17

intestinal lesions (31). Thus we propose that cathepsin D might be targeted during MTX

treatment to limit intestinal damage.

Proteasome modifications deserve discussions. In our previous study, we observed that

proteasome activity was decreased in MTX-treated rats compared with ad libitum rats both

during the acute and the recovery phases (26). Moreover, in the present work, a similar

decrease of proteasome activity in pair fed and MTX treated rats compared with ad libitum

rats at D4 was shown. At D7, these activities increased in pair-fed rats but not in MTX-treated

rats. These data indicate that nutritional starvation, which is allowed by the decrease of food

intake until D5, reduces proteasome activity as previously suggested (35). In addition, the

increase of food intake from D5 to D7 is associated with higher proteasome activity in pair-

fed but not in MTX-treated rats. Hence we can hypothesize that either MTX directly inhibits

proteasome or there are an activation of survival or economic processes in MTX-treated

animals (39, 41).

In the present study, protein synthesis was reduced in jejunal mucosa compared with

ad libitum and pair-fed rats during the acute phase of enterocolitis and was restored at D7. To

evaluate protein synthesis rate, we used a previously reported method (16) that may influence

phenylalanine and tyrosine levels. Thus, we did not take account of phenylalanine and

tyrosine levels. All together, these data suggest that a decrease of protein synthesis and an

increase of lysosomal and Ca2+-activated pathway activities are involved in the more marked

damages observed in MTX-treated rats.

In addition to this global approach of protein metabolism, we focused our interest on

specific proteins and particularly on intestinal mucins, the major component of the mucus

layer playing a key role in the non immune gut barrier function (40). The mucous layer and

mucin production are known to be qualitatively and quantitatively altered in many situations

18

of intestinal stress, including inflammatory events (10), in case of malnutrition (37), or in

specific amino acid deprivation (14). Nevertheless, transient nutritional starvation similar to

that observed in pair fed rats in the present study had not been associated with modification of

composition of mucins in globlet cells (23). In the present study, the reduced mucosal mucin

content observed at D4 in the jejunum of MTX-treated rats compared with pair fed rats

indicates a deleterious effect of the chemotherapy treatment by itself. Together with the

impaired gene expression of the main secreted intestinal mucin MUC2, this likely alters the

protection and therefore increases the vulnerability of the jejunal epithelium. Further

investigations are required to evaluate the specific role of membrane-bound mucins (MUC3 &

4) that behave differently than MUC2. At D7, defense mechanisms took place as shown by an

increased gene expression of both secreted (MUC2) and membrane-bound (MUC3 & 4)

mucins. However, the lack of response at the protein level may reflect a post-translational

limitation of mucin synthesis. As shown by a low threonine plasma level in MTX as

compared to control rats, a reduced availability in threonine is likely occurring in MTX-

treated rats, as previously suggested in other stress situations (13, 15).

The intestinal inflammatory response appeared higher in MTX-treated rats than in

pair-fed controls since we observed an increase of the histological inflammation score. It has

been suggested that pro-inflammatory cytokines, e.g. TNFα, and IL-1β, may be involved in

the amplification phase of intestinal mucositis (28) but inflammation may be the functional

consequence of the weakened barrier function, i.e. weakened epithelial integrity (atrophy,

altered protein metabolism) and altered mucus protection. However, TNFα concentration was

the only pro-inflammatory cytokine affected in MTX-treated compared with pair-fed rats in

the present study. This could be related to the timing of mucosa sampling since the pro-

inflammatory peak may have been observed early in time after MTX treatment as reported in

irinotecan-treated rats (27). In addition, malnutrition is able to induce by itself cytokine

19

expression in intestine (29) and to enhance the production of IL-1β but not of TNFα by

monocytes (7). Our results confirms this precedent work since we observed an increased

intestinal IL-1β level in the pair fed groups at day 4 and 7, but no further modifications in the

MTX groups. Thus, IL-1β was over-expressed after the food restriction and not further

stimulated by MTX itself. These data suggest that only TNFα is involved in the

pathophysiology of MTX-induced intestinal mucositis independently of food intake reduction.

All these lesions could have metabolic implications not only at the level of the gut but

also at the systemic level as shown by the disturbances of amino acid profile in duodenum and

plasma. For instance, we observed a decrease both in duodenum and plasma for taurine and

citrulline suggesting, respectively, an increased utilization for antioxidant response or an

altered gut production. For glutamine which is increased in plasma, this potentially suggests

an increased liberation from the muscle. These data underline the need to better adapt

nutritional interventions during chemotherapy as objectives to correct metabolic alterations in

the gut but also distant from gut.

In conclusion, we show that MTX treatment alters intestinal mucosa and protein

metabolism in a specific manner with a decrease of protein synthesis, in particularly of mucin

production and an increase of proteolysis mediated by lysosomal pathway. Nutritional

starvation that is associated with the treatment due to the decrease of food intake could

enhance these modifications but numbers of the MTX induced disturbances were observed

independently of the reduction of food intake. These alterations could be targeted by a

specific nutritional intervention during chemotherapeutic treatment.

20

Acknowledgements:

The authors thank Dr Jean-Paul Morin, (INSERM U644 - Rouen), Dr Christophe

Gangneux (ESITPA Engineer School - Rouen) for facilitating the use of Mithras LB 940

and SpectraMax Gemini XS respectively. The skillful technical assistance of Brigitte

Maurer (Laboratory of Medical Biochemistry, Rouen University Hospital), of Célia Duclos

and Marianne Paresy (Laboratory of Anatomo-Pathology, Rouen University Hospital) and

of Iris Segura Roggero (Nestlé Research Center, Lausanne) is gratefully acknowledged.

21

References:

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22. Keefe DM, Gibson RJ, and Hauer-Jensen M. Gastrointestinal mucositis. Semin Oncol Nurs 20: 38-47, 2004. 23. Koninkx JF, Stemerdink AF, Mirck MH, Egberts HJ, van Dijk JE, and Mouwen JM. Histochemical changes in the composition of mucins in goblet cells during methotrexate-induced mucosal atrophy in rats. Exp Pathol 34: 125-132, 1988. 24. Konsman JP, and Dantzer R. How the immune and nervous systems interact during disease-associated anorexia. Nutrition 17: 664-668, 2001. 25. Leblond J, Hubert-Buron A, Bole-Feysot C, Ducrotte P, Dechelotte P, and Coeffier M. Regulation of proteolysis by cytokines in the human intestinal epithelial cell line HCT-8: role of IFNgamma. Biochimie 88: 759-765, 2006. 26. Leblond J, Le Pessot F, Hubert-Buron A, Duclos C, Vuichoud J, Faure M, Breuille D, Dechelotte P, and Coeffier M. Chemotherapy-induced mucositis is associated with changes in proteolytic pathways. Exp Biol Med (Maywood) 233: 219-228, 2008. 27. Logan RM, Gibson RJ, Bowen JM, Stringer AM, Sonis ST, and Keefe DM. Characterisation of mucosal changes in the alimentary tract following administration of irinotecan: implications for the pathobiology of mucositis. Cancer Chemother Pharmacol 62: 33-41, 2008. 28. Logan RM, Stringer AM, Bowen JM, Yeoh AS, Gibson RJ, Sonis ST, and Keefe DM. The role of pro-inflammatory cytokines in cancer treatment-induced alimentary tract mucositis: pathobiology, animal models and cytotoxic drugs. Cancer Treat Rev 33: 448-460, 2007. 29. Lyoumi S, Tamion F, Petit J, Dechelotte P, Dauguet C, Scotte M, Hiron M, Leplingard A, Salier JP, Daveau M, and Lebreton JP. Induction and modulation of acute-phase response by protein malnutrition in rats: comparative effect of systemic and localized inflammation on interleukin-6 and acute-phase protein synthesis. J Nutr 128: 166-174, 1998. 30. McNurlan MA, Tomkins AM, and Garlick PJ. The effect of starvation on the rate of protein synthesis in rat liver and small intestine. Biochem J 178: 373-379, 1979. 31. Menzel K, Hausmann M, Obermeier F, Schreiter K, Dunger N, Bataille F, Falk W, Scholmerich J, Herfarth H, and Rogler G. Cathepsins B, L and D in inflammatory bowel disease macrophages and potential therapeutic effects of cathepsin inhibition in vivo. Clin Exp Immunol 146: 169-180, 2006. 32. Muscaritoli M, Grieco G, Capria S, Iori AP, and Rossi Fanelli F. Nutritional and metabolic support in patients undergoing bone marrow transplantation. Am J Clin Nutr 75: 183-190, 2002. 33. Pico JL, Avila-Garavito A, and Naccache P. Mucositis: Its Occurrence, Consequences, and Treatment in the Oncology Setting. Oncologist 3: 446-451, 1998. 34. Rask C, Albertioni F, Bentzen SM, Schroeder H, and Peterson C. Clinical and pharmacokinetic risk factors for high-dose methotrexate-induced toxicity in children with acute lymphoblastic leukemia--a logistic regression analysis. Acta Oncol 37: 277-284, 1998. 35. Samuels SE, and Attaix D. Fasting does not increase mRNA levels of proteolytic systems in small intestinal mucosa of the rat. J Nutr Biochem 11: 496-499, 2000. 36. Samuels SE, Taillandier D, Aurousseau E, Cherel Y, Le Maho Y, Arnal M, and Attaix D. Gastrointestinal tract protein synthesis and mRNA levels for proteolytic systems in adult fasted rats. Am J Physiol 271: E232-238, 1996. 37. Sherman P, Forstner J, Roomi N, Khatri I, and Forstner G. Mucin depletion in the intestine of malnourished rats. Am J Physiol 248: G418-423, 1985. 38. Shintani F, Kanba S, Nakaki T, Nibuya M, Kinoshita N, Suzuki E, Yagi G, Kato R, and Asai M. Interleukin-1 beta augments release of norepinephrine, dopamine, and serotonin in the rat anterior hypothalamus. J Neurosci 13: 3574-3581, 1993. 39. Tilignac T, Temparis S, Combaret L, Taillandier D, Pouch MN, Cervek M, Cardenas DM, Le Bricon T, Debiton E, Samuels SE, Madelmont JC, and Attaix D. Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis. Cancer Res 62: 2771-2777, 2002. 40. Van Klinken BJ, Dekker J, Buller HA, and Einerhand AW. Mucin gene structure and expression: protection vs. adhesion. Am J Physiol 269: G613-627, 1995. 41. Voorhees PM, Dees EC, O'Neil B, and Orlowski RZ. The proteasome as a target for cancer therapy. Clin Cancer Res 9: 6316-6325, 2003. 42. Welsh FK, Farmery SM, MacLennan K, Sheridan MB, Barclay GR, Guillou PJ, and Reynolds JV. Gut barrier function in malnourished patients. Gut 42: 396-401, 1998.

23

Figure Legends:

Figure 1: Enrichment kinetics in different pools.

Enrichment (means ± sem, mol %excess) measured in intra-tissular free amino acid (A, open

circles) or plasma (A, closed triangles) precursor pools and in mucosal proteins (B). Rats were

injected intravenously with 1500 µmol kg-1 of L-[ring-2H5]-phenylalanine and killed after 5,

10, 15 or 20 minutes (n=3 at each time).

Figure 2: Body weight changes

Evolution of body weight in control ad-libitum (open circles) and pair-fed (open triangles)

and in MTX-treated rats (closed triangles). Values are means ± sem from 12 (control ad-

libitum), 24 (control pair-fed) and 24 rats (MTX) from D0 to D4 and from 12 (control ad-

libitum), 12 (control pair-fed) and 12 rats (MTX) from D5 to D7. P values: Treatment,

p<0.0001; Day, p<0.0001; Interaction, p<0.0001. Values without a common letter differ

(p<0.05).

Figure 3: Histology, villus height and histological scores in the jejunum of rats.

(A) Jejunal mucosa observation (X 5) in control ad libitum rats (1), pair-fed rats at D4 (2), in

MTX-treated rats at D4 (3) and D7 (4). (B) Villus height and (C) inflammation score in

jejunal mucosa of control ad libitum rats (grey bars) and pair-fed (open bars) and MTX-

treated rats (closed bars) at D4 and D7. Values are means ± sem. P values: (B) Treatment,

p<0.0001, Day, p<0.0001; Interaction, p=0.0008. (C) Treatment, p<0.0001, Day, NS;

Interaction, NS. *, p<0.05 versus control ad libitum rats. +, p<0.05 versus control pair-fed

rats. †, p<0.05 versus D4.

Figure 4: Fold change of mucin mRNA expression in the jejunum of rats.

24

Real-Time PCR analysis of the expression of MUC2, 3 and 4 mRNA in the jejunum of MTX-

treated rats at D4 (open bars) or D7 (grey bars). Values are fold change versus control pair-

fed rats and expressed as means (lower and upper confidence limit). *, p<0.05; **, p<0.01;

***, p<0.001 vs control pair fed rats using rejection test.

Figure 5: Proinflammatory cytokine concentrations in jejunal mucosa of rats.

(A) TNFα and (B) IL-1β jejunal concentrations in control ad libitum rats (grey bars) and pair-

fed (open bars) and MTX-treated (closed bars) rats at D4 and D7 (means ± sem). Two-way

ANOVA P values: (A, TNFα) Treatment, p<0.0001; Day, NS; Interaction, NS. (B, IL-1β)

Treatment, p<0.0001; Day, NS; Interaction, NS. * p<0.05 versus control ad libitum rats. +

p<0.05 versus control pair fed rats.

Figure 6: Protein content and synthesis in the jejunum of rats.

Protein content (A) and FSR (B) was evaluated in jejunal mucosa of control ad libitum rats

(grey bars) and pair-fed (open bars) and MTX-treated (closed bars) rats at D4 and D7 (means

± SD). P values: (A) Treatment, p<0.001, Day, NS; Interaction, NS. (B) Treatment, NS, Day,

p=0.0003007; Interaction, p=0.002. *, p<0.05 versus control ad libitum rats. + p<0.05 versus

control pair fed rats. †, p<0.05 versus D4.

Figure 7: Proteolytic pathway activities in the jejunal mucosa of rats.

(A) Cathepsin D, (B) calpain and (C) peptidyl-glutamyl peptidase, trypsin- and chymotrypsin-

like proteasome activities in jejunal mucosa of control ad libitum rats (grey bars) and pair-fed

(open bars) and MTX-treated (closed bars) rats at D4 and D7. Values are means ± sem. P

values: (A) Treatment, p<0.0001, Day, p=0.0182; Interaction, p=0.0043. (B) Treatment, NS,

25

Day, NS; Interaction, p=0.011. (C) Treatment, p<0.001, Day, NS; Interaction, NS. *, p<0.05

versus control ad libitum rats. + p<0.05 versus control pair fed rats. †, p<0.05 versus D4.

Figure 8: Correlation between plasma citrulline concentration and villus heights.

Plasma citrulline concentration of control ad libitum rats (open lozenge), of pair-fed rats at

D4 (closed triangles) or at D7 (open triangles) and of MTX-treated at D4 (closed circles) or

D7 (open circles) was reported in function of villus height. (Pearson correlation, r=0.677,

p<0.0001).

26

Table 1: BrdU incorporation, GSH content and Mucin contents in jejunal samples in control pair-fed and MTX-treated rats.

Values are means ± SD. *, p<0.05 *** p<0.001 versus control pair-fed rats and † p<0.001 versus D4 using Bonferroni posttests.

Parameters

Control pair-fed rats MTX-treated rats P values

D4 D7 D4 D7 Treatment Day Interaction

BrdU incorporation 1.7 ± 0.8 2.4 ± 1.0 1.3 ± 0.6 2.0 ± 0.5 NS p=0.0031 NS

GSH

(µmol / g tissue) 3.43 ± 1.36 4.78 ± 1.89 3.31 ± 0.83 5.27 ± 0.56 NS p=0.0003 NS

Mucins

(µg / g tissue) 2.76 ± 0.73 3.00 ± 0.70 2.08 ± 0.73 1.90 ± 0.66 p=0.0002 NS NS

27

Table 2: Selected plasma amino acid concentrations (µmol/L) in control and MTX-treated rats at D4 and D7.

ad libitum rats pair-fed rats MTX-treated rats P values

D7 D4 D7 D4 D7 Treatment Day Interaction

ALA 645 ± 26 562 ± 17 729 ± 24 † 615 ± 21 1182 ± 21 *,+, † p<0.0001 p<0.0001 p<0.0001

ASN 56 ± 4 75 ± 13 * 89 ± 21 * 84 ± 14 * 78 ± 33 * p<0.0001 NS NS

CIT 110 ± 3 79 ± 9 * 95 ± 51 14 ± 3 *,+ 39 ± 35 *,+ p<0.0001 NS NS

GLU 292 ± 20 191 ± 14 226 ± 89 140 ± 22 * 207 ± 38 p=0.0021 NS NS

GLY 203 ± 13 475 ± 12 * 474 ± 15 * 353 ± 12 *,+ 248 ± 37 +, † p=0.0022 p=0.0220 p=0.0078

HIS 68 ± 3 91 ± 16 * 105 ± 29 * 93 ± 10 * 115 ± 21 *, † p<0.0001 p=0.0128 NS

ILE 131 ± 4 123 ± 13 112 ± 14 104 ± 18 *,+ 116 ± 27 p=0.0064 NS NS

PRO 265 ± 16 153 ± 8 * 190 ± 30 * 143 ± 16 * 380 ± 29 *,+, † p<0.0001 p<0.0001 p<0.0001

SER 247 ± 11 391 ± 9 * 411 ± 14 * 313 ± 8 *,+ 348 ± 18 *,+ p<0.0001 NS NS

THR 386 ± 21 373 ± 10 400 ± 11 297 ± 10 *,+ 358 ± 21 † p=0.0005 p=0.0319 NS

VAL 310 ± 11 217 ± 13 * 229 ± 20 * 226 ± 20 * 276 ± 30 p<0.0001 NS NS

Values are means ± SD. * p<0.05 vs. control ad libitum rats, + p<0.05 vs control pair-fed rats and † p<0.05 vs D4.

28

Table 3: Selected duodenal free amino acid concentrations (µmol/L) in control and MTX-treated rats at D4 and D7.

Control ad libitum rats

Control pair-fed rats MTX-treated rats P values

D4 D7 D4 D7 Treatment Day Interaction

ALA 5999 ± 371 2612 ± 66 * 3622 ± 256 *, † 2181 ± 82 * 6186 ± 308 *,+ P<0.0001 P<0.0001 P<0.0001

ARG 228 ± 17 171 ± 9 171 ± 8 255 ± 34 + 255 ± 31 + P=0.0014 NS NS

ASP 2936 ± 114 1654 ± 66 * 2373 ± 106 *, † 1862 ± 60 * 2324 ± 180 *, † P<0.0001 P<0.0001 P<0.0001

CIT 633 ± 29 313 ± 12 * 349 ± 17 * 74 ± 3 *,+ 149 ± 20 *,+ P<0.0001 NS NS

GLN 601 ± 34 606 ± 30 604 ± 48 1160 ± 75 *,+ 983 ± 87 *,+ P<0.0001 NS NS

GLU 5632 ± 132 3619 ± 162 * 4115 ± 15 * 4894 ± 219 *,+ 5066 ± 343 + P<0.0001 NS NS

GLY 2101 ± 90 2317 ± 96 2590 ± 133 * 2536 ± 133 * 1677 ± 125 *,+, † P=0.0037 P=0.0449 P<0.0001

HIS 145 ± 10 130 ± 8 142 ± 7 148 ± 10 174 ± 10 P=0.0375 NS NS

ILE 289 ± 21 179 ± 9 * 164 ± 13 * 165 ± 15 * 163 ± 12 * P<0.0001 NS NS

LEU 602 ± 41 386 ± 17 * 384 ± 25 * 422 ± 34 * 435 ± 26 * P<0.0001 NS NS

LYS 595 ± 35 409 ± 16 * 401 ± 14 * 498 ± 34 * 516 ± 37 + P<0.0001 NS NS

MET 162 ± 13 136 ± 8 107 ± 4 * 135 ± 8 137 ± 12 P=0.0006 NS NS

ORN 155 ± 7 104 ± 4 * 109 ± 6 * 96 ± 9 * 91 ± 10 * P<0.0001 NS NS

PRO 1180 ± 78 494 ± 21 * 426 ± 17 * 329 ± 18 * 830 ± 63 *,+, † P<0.0001 P=0.0017 P<0.0001

SER 737 ± 34 750 ± 28 783 ± 45 787 ± 30 732 ± 51 NS NS NS

29

TAU 5376 ± 334 8829 ± 266 8320 ± 365 *, † 4808 ± 255 *,+ 7461 ± 494 * P<0.0001 P<0.0001 P<0.0001

THR 927 ± 45 591 ± 17 * 647 ± 34 * 589 ± 28 * 661 ± 42 * P<0.0001 NS NS

VAL 576 ± 34 310 ± 13 * 301 ± 22 * 305 ± 19 * 355 ± 26 * P<0.0001 NS NS

Values are means ± SEM. *, p<0.05, **, p<0.01 and ***, p<0.001 vs control pair-fed rats. † p<0.01 and ‡ p<0.001 vs D4

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