Kelly Connaughton

Journal of Undergraduate Research
Volume 4, Issue 11 - August 2003

Effects of Dietary Supplementation with RNA on Recovery of Intestinal Function after Administration of Methotrexate

INTRODUCTION

In the healthy body, cells in virtually all tissues and organs are constantly maturing and dividing to replace old and damaged cells in order to maintain a healthy body. In a cancer patient, however, some cells keep dividing without proper control, which results in abnormal new cell growth (neoplasm) and replication (8).

Treatment for the person with cancer typically involves a combination of surgery coupled with radiation and/or chemical therapy. Chemotherapy involves the use of one or several antineoplastic (anticancer) drugs to kill cancer cells. Methotrexate is an example of an anticancer drug that interferes with cellular reproduction and is also used in the treatment of psoriasis and certain inflammatory diseases (6). While these kinds of drugs are toxic to cancer cells, many are also toxic to healthy cells and thereby give rise to many side effects. The main areas of the body that may be affected by chemotherapy are those where normal cells rapidly divide and grow, such as the lining of the mouth, the digestive system, as well as skin, hair and bone marrow. These drugs also reduce resistance to infections and decrease immune function. These side effects may be exacerbated in people already suffering malnutrition as a consequence of the cancer (8).

Since the body can synthesize nucleotides or ribonucleic acid (RNA), they are not considered to be dietary essential nutrients. Nucleotides are involved in several biochemical and physiological processes that are fundamental to cellular activity. They act as precursors of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the carrier of genetic information and also serves as a template for the formation of RNA (7) Most RNA molecules, in turn, direct synthesis of cellular proteins. Under certain physiological stresses, the synthesis of nucleotides may not be rapid enough to meet the body’s requirement for tissue repair and immune function. It is thought that a dietary supplement of RNA may promote maintenance and recovery of rapidly proliferating cells such as those of the epithelial tissues after the body is exposed to a chemical stress such as methotrexate (4)

METHODS

Animals and Diet Treatment

Male ICR mice weighing 26.6g (about 4 weeks of age from Harlan Teklad, Madison, WI) were housed four to a cage in a temperature-controlled facility with a constant 12-hour light-dark cycle. Mice were allowed to acclimate to the laboratory for a few days prior to the experiment when then they were first fed ad libitum, a 20% casein-based diet for sixteen days. (Casein is very low in naturally occurring nucleotides and is considered to be essentially nucleotide-free (4).) The mice were then placed on a protein diet for six days to induce mild protein malnutrition to simulate a nutritional stress frequently seen in chemotherapy patients. One-half of the mice then received a single intraperitoneal injection of methotrexate at 35-mg/kg-body weight. This dose is known to induce a temporary decrease in appetite. Finally, for the last six days, all mice were fed the casein-based diet with or without supplemental purified yeast RNA at 1.5%. After six days on the protein-containing diet, the mice were killed and the small intestine was rapidly cut into three equal lengths, denoted as proximal, mid, and distal. The segments were rinsed through with cold saline and frozen at -20Ö C pending analysis.

Tissue Preparation

After thawing and maintaining the tissue on ice, each segment was homogenized at 20% weight/volume in 0.9%saline with 0.5% protease inhibitor.

Assays

Enzyme activities were assessed for the disaccharidases sucrase and lactase, and alkaline phosphatase. The concentrations of DNA, RNA, and total protein were also measured.

The disaccharidase assays consisted of a two-step procedure. First, the samples were incubated with lactose or sucrose in buffer. Then, hydrolyzed substrate was determined using 2’-Azinobis(3-ethylbenzthiazoline) sulfonic acid (ABTS) which produces a blue-green color. The intensity of the color is proportional to the amount of hydrolyzed substrate, which indirectly measures the activity of the lactase or sucrase enzyme (1).

To measure alkaline phospahtase enzyme activity, a substrate buffer consisting of p-Nitrophenylphosphate (PNPP) was added to the samples and incubated. Alkaline phosphatase hydrolyzes the synthetic substrate PNPP to free p-nitrophenol. The activity of the enzyme was determined colorimetrically; the substrate was colorless, while the product was yellow. The intensity of the color is proportional to the concentration of the product, which indirectly measure the activity of the enzyme (3)

DNA and RNA determination were measured using the Burton Method and Orcinol Method, respectively ( 9,5 ).

Total protein determination was measured using the Biuret assay ( 2 ).

Statistical Analysis

Enzyme activities and determination of DNA, RNA, and protein concentrations for each group was analyzed using analysis of variance using Statistical Analysis Systems (SAS Institute, Cary, NC). Data are expressed as means + standard error of the mean, and was at least significance difference at p ≤ .05.

RESULTS

Figure 1. Changes in Body Weight

Figure 1 represents the diet treatment that was fed to the mice for 28 days and their resulting change in body weight. According to the figure, after six days of feeding on the protein-free diet, all groups lost approximately 10% of their body weight. After the mice were repleted with the casein-based diet with or without purified yeast RNA; all groups regained all weight except for the group that was treated with MTX+RNA which continued to lose weight.

Figure 2. Protein Concentration.

According to Figure 2, the mean protein concentration in the distal region was slightly higher in all treatment when compared to the control, however, only the group containing MTX was significant. There was no significant difference among the proximal and mid groups.

Figure 3. RNA Concentrations.

Figure 4. DNA Concentrations.

There was no significant difference in any of the groups for RNA (Figure 3). The RNA group of the distal segment was lower than the other groups and when compared to the proximal and distal segments in DNA. The DNA content was not affected in the proximal or distal region (Figure 4).

Figure 5. Lactase Activity

Figure 6. Sucrase Activity

When measuring the enzyme activity of the disaccharidases, the group injected with MTX and also consuming the RNA supplemented diet showed a decrease in sucrase activity in the proximal segment and a decline of 62% in the mid region when compared to the mice who were also injected with MTX but fed the casein diet (Figure 5). A decrease in lactase activity was reported again in the MTX +RNA group for the proximal and mid regions. The proximal area was reduced by 51% and the mid decreased by 68%.

Figure 7. Alkaline Phosphatase Activity.

No significant differences were found in alkaline phosphatase activity (Figure 7).

CONCLUSION

It was hypothesized that an RNA supplemented diet fed to mice that were administered the anticancer drug methotrexate would promote maintenance and recovery of rapidly proliferating cells (i.e. epithelial tissue). Under normal conditions, dietary nucleotides are not considered to be essential for support and growth. In a stressed state, an accelerated proliferation of tissues with short-lives is required. It was been shown that an exogenous supply of nucleotides in the form of intact RNA would be beneficial.

Methotrexate, a common anticancer drug, reduces the half-life of mucosal epithelial cells and is associated with a decline in digestive-absorptive function. I studied the effects of adding RNA to the diet on the response of the small intestine of mice that were mildly protein malnourished and injected with MTX.

The addition of RNA to a casein-based (nucleotide-free) diet in protein-malnourished mice not given MTX (group RNA) did not affect body weight gain or small intestinal tissue levels of protein, DNA and RNA, and digestive function as examined by measuring disaccharidase and alkaline phosphatase activity. Mice who were on the casein-based diet and given a dose of MTX (group MTX) showed no unusual effects six days after injection when compared with the control mice. The six-day period was a sufficient amount of time to allow the gut to completely recover without the need for dietary supplementation.

Compared to the other groups studied, group MTX+RNA fared poorly. This group lost weight in the protein-repletion period and did not recover disaccharidase activity. Thus, the addition of RNA at 1.5% of the diet appeared to delay recovery of the gut from MTX administration.

REFERENCES

Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem 7:18-25,1964
Doumas BT, Bayse DD, Carter RJ, et al: A candidate reference method for determination of total protein in serum I. Development and validation. Clin Chem 27: 1642-1650, 1981.
Eichholz A: Structural and functional organization of the brush border of intestinal epithelial cells. 3. Enzymatic activities and chemical composition of various fractions of tri-disrupted brush borders. Biochem Biophys Acta 135:475-482, 1967.
Lin, Cheng-mao. Effect of Dietary Nucleotide Supplementation on In Vivo and In Vitro Immune Function in Protein-Malnourished Mice. University of Florida. Ph.D. dissertation. December 1995.
Lin RI, Schjeide OA. Micro estimation of RNA by the cupric ion catalyzed orcinol reaction. Anal Biochem 27:473, 1969.

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