Journal of Undergraduate Research
Volume 4, Issue 12 - September 2003

Dietary RNA Supplementation Increases Signs of Intestinal Inflammation Following Administration of an Anticancer Drug

Michelle Weiner

ABSTRACT

Methotrexate (MTX), an anticancer drug causes side effects such as destroying rapidly dividing normal cells. When rapidly dividing intestinal cells are destroyed, the integrity of the mucosal barrier may be compromised thereby resulting in the production of inflammatory mediators that further damage the intestinal epithelial barrier. Ribonucleic acid (RNA) is supplemented in the diet to aid in regeneration of new cells. Therefore, it is hypothesized that RNA supplementation will be beneficial in mice treated with MTX. One-month-old mice were divided in four diet/treatment groups, fed a casein-based nucleotide-free diet for 16 days, and then given a protein free diet for six days. After 22 days, groups were given an intraperitoneal injection of MTX at 35mg/kg body weight with or without RNA nucleotides supplemented at 1.5% of the diet for 6 days. The small intestines were obtained, divided into three portions: proximal, medial, and distal and assayed for protein content as well as myeloperoxidase (MPO) activity as an indirect indicator of neutrophil infiltration, i.e. inflammation. Average body weight of the (+)RNA(+)MTX group was significantly lower than the three other groups with a P ≤ 0.001 and MPO activity in both the proximal and medial sections of (+)RNA(+)MTX group was significantly higher with a P ≤ 0.05. These results suggest that supplementation of RNA coupled with MTX increases neutrophil infiltration and/or causes an inflammatory response that may further damage the intestine.

INTRODUCTION

Cancer patients are often treated with anticancer drugs such as methotrexate (MTX). Methotrexate is a folic acid antagonist and a competitive inhibitor of dihydrofolate reductase that blocks deoxyribonucleic acid (DNA) synthesis and cell replication (4). Therefore, this anticancer drug destroys rapidly dividing cells such as cancer and intestinal cells. When the intestinal cells are damaged by certain stresses such as anticancer drugs, the intestinal barrier is broken down. This allows the translocation of toxins and bacteria from the intestinal lumen precipitating inflammatory and immune responses (1).

Nucleotides are not considered to be essential dietary components for normal individuals (3). Nucleotides are synthesized in the intestine from dietary amino acid precursors when a nucleotide-free diet is consumed (7). During times of stress, the synthesis of ribonucleic acid (RNA) may fall below desired demand. If RNA cannot be synthesized in sufficient quantities to support the regeneration of these epithelial cells, the barrier between the lumen of the intestine and the internal environment may be lost. Therefore, the addition of RNA to an RNA-free diet may, in theory, support more rapid intestinal cell regeneration during times of increased cell turnover. The purpose of this experiment was to determine whether an RNA-supplemented diet will reduce intestinal inflammation in mice treated with MTX.

MATERIALS AND METHODS

Animals and Diets

Thirty two male 4-week old ICR male mice (Harlan Teklad, Madison, WI) were caged in groups of four in a temperature controlled room with a 12-hour light-dark cycle at the University of Florida and allowed 5-7 days to acclimate before the study began. During this period, the mice were fed a stock diet. The mice were then randomly assigned to one of four groups (Table 1). Each group was fed a casein-based nucleotide-free diet for 16 days and then placed on a protein-free diet for six days. The short period on the protein-free diet was designed to induce a state of mild malnutrition, as commonly encountered in patients with cancer who undergo chemotherapy. After 22 days, groups were given an intraperitoneal injection of MTX at 35mg/kg body weight with or without intact RNA (isolated from yeast) supplemented at 1.5% of the casein-based diet. The dose of MTX was selected to be therapeutic and no animals succumbed to the injection.

Table 1
Experimental Design
Group Diet for 16 days Diet for 6 days Diet and treatment for final 6 days
(+)MTX(-)RNA Casein Protein-Free Casein + MTX
(-)MTX(-)RNA Casein Protein-Free Casein
(+)MTX(+)RNA Casein Protein-Free Casein + RNA + MTX
(-)MTX(+)RNA Casein Protein-Free Casein + RNA

 

Six days later, the mice were fully anesthetized and exsanguinated from the inferior vena cava. The small intestines were harvested and weighed. The tissues were then divided into three equal portions: proximal, medial, and distal. The portions of the small intestine were stored at –80÷C until assayed. Tissues were subsequently thawed and weighed again and assayed for myeloperoxidase (MPO) using an adaptation of a previously described method (2). Mice were weighed prior to each diet and/or treatment change and at the end of the experiment. All procedures were approved by the University of Florida Animal Care and Use Committee.

Tissue Preparation

Tissues of the small intestine were homogenized at 20% weight/volume in 20 mM potassium phosphate dibasic (KH2P04) with 1 mM ethylene diamine-tetraacetic acid (EDTA), pH 7.4. An aliquot (50µl) of each sample was removed and refrigerated for subsequent protein assay. Protein content of the proximal, medial, and distal small intestines was determined using the Bio-Rad Protein Assay Kit (Hercules, CA). The remaining homogenate was then brought up to 1.5 times its new volume with KH2P04 with 1 mM EDTA and centrifuged at 12,000 x g for 20 minutes. The supernatant was removed and the pellet was reconstituted to a final volume of 1 ml with 50 mM of acetic acid with 0.5% hexadecyltrimethyl ammonium hydroxide (HETAH) at pH 6.0. The remaining sample was vortexed, re-homogenized, and sonicated using the Sonic Dismembrator (Fisher Scientific, Pittsburgh) for 30 seconds and submitted to two cycles of freezing and thawing. The sample was centrifuged at 12,000 x g for 20 minutes and the supernatant was removed for assay.

MPO Assay

The measurement of MPO activity in tissue is a convenient and accurate means of assessing the number of neutrophils at an inflammatory site (5). Myeloperoxidase activity was determined by measuring the hydrogen peroxide-dependent oxidation of 3,3’,5,5’,-tetramethylbenzidine (TMB) (2). This produced a green-blue color change at a wavelength of 655 nm. The supernatant of each sample was added to a reaction buffer containing 315 µl 0.8 M KH2P04 (pH 5.4) to which 25 µl of 10% HETAH, and 50 µl of 16 mM TMB in dimethylformamide. The samples were immersed in a shaking water bath at 37÷C for five minutes. The reaction was initiated by adding 10 µl of 30 mM hydrogen peroxide and incubated for three minutes. The reaction was terminated by the sequential addition of 10 µl of catalase (300 µg/ml) and 2 ml of 0.2 M sodium acetate was added. The mixture was poured into cuvettes and the absorbance was measured at 655 nm on a spectrophotometer (Beckman DU640, Irvine, CA). Units of MPO activity were calculated from a standard curve that used peroxidase enzyme (Sigma) as the standard enzyme. Myeloperoxidase data are expressed as U/min/g protein. One unit of peroxidase produces 1.0 mg purpurogallin from pyrogallol in 20 seconds at ph 6.0 at 20÷C.

Statistical Analysis

Myeloperoxidase activity for treatment with or without methotrexate and/or RNA for each group was analyzed using a 2 way analysis of variance using Statistical Analysis Systems (version 8.2, SAS Institute, Cary, NC). Data are expressed as means +/- standard error of the mean, and a P ≤0.05 denotes significance.

RESULTS

The weight of all the mice increased after given the casein diet and was slightly lower after the protein-free diet. However, during the final six days, the weight of the (+)MTX(+)RNA group (28.1 + 1.15g) was significantly lower (P ≤ 0.001) than
(-)RNA(-)MTX (33.5 ± 0.876), (-)RNA(+)MTX (33.7 ± 0.713) and (+)RNA(-)MTX (35.4 + 0.759).

Myeloperoxidase activity was significantly higher in (+)MTX(+)RNA compared to the (+)MTX(-)RNA groups in both the proximal and medial intestines (Figure 1). Myeloperoxidase activity in the proximal section for (+)MTX(+)RNA showed 8.4 times more activity than (+)MTX(-)RNA and the medial (+)MTX(+)RNA showed 5 times more MPO activity than (+)MTX(-)RNA. MPO activity was also significantly greater in both the proximal and medial sections in (+)MTX(+)RNA compared to the (-)MTX(+)RNA. Group (+)MTX(+)RNA illustrated approximately 4.3 and 2.2 times greater MPO activity than in group (-)MTX(+)RNA respectively for the proximal and medial sections. There were no significant differences in MPO activity in the distal section among any groups.

Figure 1. Myeloperoxidase (MPO) Activity of the Proximal Medial and Distal Portions of the Small Intestines in Mice

Figure 1. Myeloperoxidase (MPO) Activity of the Proximal, Medial and Distal Portions of the Small Intestines in Mice (+/-) Ribonucleic acid (RNA) and (+/-) Methotrexate (MTX). N= 7-8 mice/ group. * P ≤ 0.05 vs. all other groups.

DISCUSSION

The present study indicates that an RNA-supplemented diet does not reduce intestinal inflammation in mice administered the common anticancer drug MTX. In fact, the combination of MTX and RNA resulted in significantly higher MPO activity in the proximal and medial intestinal sections and significantly lower final body weights. This suggests increased neutrophil infiltration or an increased inflammatory response in the intestine of (+)MTX(+)RNA mice.

Dietary supplementation of RNA without MTX treatment had no effect on MPO activity. Therefore, dietary supplementation of RNA by itself does not appear to induce intestinal inflammation. However, the combination of MTX and RNA may have aggravated MTX-induced inflammation resulting in a slowed or delayed recovery. When stimulated neutrophils infiltrate into a site they initiate a series of reactions that produce oxidizing agents and release enzymes that can be damaging to the surrounding tissue (2). Additionally, MPO, a principal component found within neutrophil granules reacts with hydrogen peroxide and chloride ion to produce hypochlorous acid and free chlorine (6). Hypochlorous acid is a powerful oxidizing agent that can attack a wide range of biomolecules and thereby induce cell death. Therefore, stimulated neutrophils can cause additional damage to inflamed tissues.

While the combination of MTX and RNA increased MPO activity, there was no difference in MPO activity and final average body weights between (-)MTX(-)RNA and (+)MTX(-)RNA groups. This suggests that six days after MTX was injected, the inflammatory response had subsided and the (+)MTX(-)RNA mice had recovered.

In the distal section of the small intestine, the combination of MTX and RNA did not appear to have a detrimental effect. It is possible that RNA was absorbed prior to reaching the distal intestine and was therefore not present or not present in great enough quantities to aggravate MTX-induced injury. The increase in MPO activity from the proximal to distal section of the small intestine parallels the increasing gradient in intestinal bacterial counts (1). As a result of the greater exposure to bacteria and antigens, more neutrophils may be present leading to the higher MPO activity observed in the distal intestinal section of all groups (1).

In conclusion, this study showed that an RNA-supplemented diet was not beneficial in mice administered MTX. As a result of increased intestinal MPO activity and a decrease in body weight in (+)MTX(+)RNA mice, the RNA was in fact, detrimental. Because RNA is added to nutritional formulas given to patients who have cancer or who are critically ill, future studies are needed to determine the mechanisms for this detrimental effect.

REFERENCES

  1. Gebbers J, Laissue J. Immunologic Structures and Functions of the Gut. Schweiz.Arch. Tierheilk. 131: 221-238, 1989.

  2. Grisham M, Benoit J, Granger D. Assessment of Leukocyte Involvement during Ischemia and Reperfusion of Intestine. Am. J. Physiol. 251.G567, 1986.

  3. Grimble G, Olwyn W. Nucleotides. Nutrition and Immunology: Principles and Practice. Humana Press, Inc., NJ.

  4. Lederle, Mylan. Methotrexate. Clinical Pharmacology, 2000.

  5. Ormrod D, Harrison G, Miller T. Inhibition of Neutrophil Myeloperoxidase Activity by Selected Tissues. Journal of Pharmalogical Methods. 137-142, 1987.

  7. Nunez M, Ayudarte M, Morales D, Suarez M, Gil A. Effect of Dietary Nucleotides on Intestinal Repair in Rats with Experimental Chronic Diarrhea. Journal of Parental and Enteral Nutrition. 14: No.6, 1990.

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