Fluorouracil Plus Leucovorin Induces Submandibular Salivary Gland Enlargement in Rats

Abstract

The administration of 5-fluorouracil (FU) and leucovorin (LV) to rats induced a previously unreported sialoadenitis-like toxicity. Four different treatment regimens were used: daily-times-5 iv or ip injections of LV (200 mg/kg) followed 30 minutes later by FU (27.5 mg/kg or 35 mg/kg). These treatments resulted in 3 severity levels of systemic toxicity indicated by changes in body weight. In addition to the well known FU+LV-induced diarrhea, myelosuppression, and stomatitis, facial edema, and enlargement of the submandibular salivary gland were consistently seen. Facial edema occurred almost exclusively in rats that subsequently underwent excessive weight loss and were euthanized. The submandibular, but not parotid or sublingual, salivary gland was enlarged and the severity of this effect changed in a bell-shaped relationship with respect to increasing FU+LV induced loss of body weight. Histologic examination of affected glands established the occurrence of bacterial infection, sialoadenitis and destruction of gland tissue. This paper provides the first known documentation of FU+LV treatment-induced selective pathology of the submandibular salivary gland. The selectivity of this toxicity, apparently not normally seen in humans, to the submandibular salivary gland of the rat is of interest and its mechanism warrants further investigation.

Introduction

5-Fluorouracil (FU) is a synthetic analog of uracil that is metabolized through the same enzyme pathways that act on uracil. Like uracil, FU is metabolized at many steps along these pathways, and inhibits the enzyme thymidylate synthase (TS) and thus disrupts the production of thymidine, resulting in decreased DNA synthesis. FU incorporation into DNA and RNA cause disruption of their normal functions, which along with decreased DNA synthesis results in cell death. Inhibition of TS by FU occurs after the formation of a tertiary complex of FU, 5,10- methylenetetrahydrofolate (CH₂-FH₄) and TS (Heidelberger, 1965; Calabresi and Chabner, 1990). The noncytotoxic agent leucovorin (LV), increases the pool of tetrahydrofolates, increasing the formation of the tertiary complex and enhancing the inhibition of TS (Evans et al., 1981). Many drug combinations used to treat invasive cancers (e.g., metastatic breast adenocarcinoma) include FU. Colorectal carcinoma is usually treated with a regimen that includes FU in combination with LV. FU+LV combination treatment has been shown to result in a 20% complete response rate and increases the rate of survival up to 62% to 71% at 3 years (Madajewicz et al., 1984; O’Connell, 1989; Mini et al., 1990; Marsoni, 1995). The liver is the main organ where FU metabolism occurs, with some seen in the lungs and kidney (MacMillan et al., 1978). Between 20% and 50% of FU can be catabolized during the first pass through the liver (Jones et al., 1978; Diasio and Harris, 1989). Therefore, intravenous (iv) administration is best to achieve high peak blood levels, with intraperitoneal (ip) administration leading to lower peak blood levels. Intraperitoneal administration however, has been shown to result in higher exposure of the liver to FU+LV, the main site of colorectal carcinoma metastasis (Jones et al., 1978). Although the main mode of FU administration in the clinic is iv, several studies with colon and ovarian cancer patients have examined the use of ip administration to achieve high drug levels in the liver and peritoneum without the causing severe systemic toxicity (Sugarbaker et al., 1985; Campora, 1987; Bruckner, 1988; Kuzuya et al., 1994). Although the dose dependency is different, the toxicity profile caused by FU+LV is the same if administered iv or ip. The main dose-limiting toxicities include diarrhea, myelosuppression, and stomatitis (Cunningham, 1984; Madajewicz et al., 1984; O’Connell, 1989; Calabresi and Chabner, 1990). Other FU+LV-induced toxicities, with a reported low incidence include: hand foot syndrome, alopecia, nausea, vomiting, dermatitis, cardiac toxicities, and decreased salivary gland secretion (Celio et al., 1983; Lokich and Moore, 1984; Slavik et al., 1993; Ramos et al., 1996; Joulia et al., 1999).

Cao and Rustum (1998), had reported on the ability of IL-15 to protect against FU+LV induced toxicity and potentiate the FU+LV mediated antitumor activity. Our group was interested in the possible involvement of the host immune system in these effects; however, due to a lack of an adequate supply of IL-15, this was never completely explored and are not discussed herein. Nevertheless, in the initial control experiments, using variants of a daily treatment regimen studied by Cao and Rustum (1998), 2 toxicities, facial edema and submandibular salivary gland enlargement, were observed in rats. A search of the literature indicated that these 2 toxicities had not been previously reported. Therefore, a description of these toxicities and a possible etiology are discussed here.

Materials and Methods

Rats

Female Fisher F344/NHsd rats were obtained from Harlan Sprague–Dawley, Indianapolis, IN. Rats were kept on corn-cob bedding in sterilized filter top cages with controlled humidity, 12-hour day/night cycles at 72°C; they were fed sterilized LM-485 rodent chow containing 5% fat from Harlan Teklad, Madison, WI, and reverse osmosis water was provided ad libitum. Experiments were started when rats were 8-to 12-weeks old. The rats were housed both prior to and during the experimental procedures under a specific pathogen-free barrier system. The integrity of the barrier system is routinely monitored by a sentinel animal test system, based on this testing, it was verified that it was maintained throughout the experimental procedures. All studies were conducted according to IACUC approved protocols. Animals were monitored daily and were euthanized as necessary to avoid unnecessary pain and suffering. All applicable criteria for the humane treatment and care of animals given in the “Guide,” and the Animal Welfare Act/Regulations were observed.

Agents

FU was obtained from American Pharmaceutical Partners Inc., Los Angeles, CA. LV calcium was obtained from Bed-ford Laboratories, Bedford, OH, or from Immunex, Seattle, WA.

Drug Injections and Blood Collection

Intravenous procedures were all performed while rats were restrained with a Universal Rat Restrainer (700R) from Brain-tree Scientific, Braintree, MA. Rat tails were immersed in 50°C water for 30 to 40 seconds. Intravenous access was gained in the tail using a Safelet over the needle (27 ga) catheter (24 ga) from EXEL Int., Culver City, CA. A new catheter was inserted for each drug injection and for each blood collection. Drug injections were also given ip with a 30-gauge ½″ needle. Rats were injected daily for 5 days with LV (200 mg/kg) followed 30 minutes later by either a high dose (35 mg/kg) or low dose of FU (27.5 mg/kg).

For absolute blood cell counts, blood was collected using a 40 μl end-to-end micropipette and placed into a Haema-Line 2 Reagent Silo both from Delta Scientific, Ivyland, PA. Absolute blood cell counts were determined using a System 9000 Hematology Series Cell Counter from Baker Instruments, Allentown, PA, which measured white blood cells, red blood cells, hemoglobin concentration, hematocrit, mean red blood cell volume, mean concentration of hemoglobin per red blood cell, and platelets. For differential blood cell counts, blood was collected in a capillary tube and 2 blood smears per rat were made on microscope slides. The slides were dried, fixed, and stained with a modified methylene blue-eosin stain using a Hema-TEK 2000 from Miles Inc., Elkheart, IN. Differential white blood cell counts were then performed on, at least, 200 leukocytes from each rat’s blood sample. The average percentage of each cell type was then multiplied by the absolute white blood cell count to give an absolute count for each white blood cell type.

Necropsy/Histologic Analysis

Rats were necropsied to examine the effects of treatment. Animals were sacrificed by CO₂ asphyxiation and their abdominal and thoracic cavities were opened and examined. The neck and mouth were also dissected to examine the submandibular and parotid salivary glands and for detection of stomatitis. Selected tissues, depending on gross findings, were taken for histologic examination. Tissues of interest were placed in formalin and given to the RPCI Preclinical Histology Facility for processing and hematoxylin/eosin staining.

Experimental Design

Toxicity was evaluated during 5 independent experiments. All doses and routes of administration were tested in 1 large experiment, while selected doses and routes were examined in the 4 smaller experiments. In each experiment, 3 rats per group were used. These experiments resulted in a total of 6 to 15 rats being tested for each treatment variable. Toxicity data expressed as loss of body weight and observable symptoms from the single comprehensive experiment with 6 rats/experimental variable are presented, but were also examined during the 4 other experiments. Toxicity expressed as myelosuppression are from a single experiment with 3 rats/treatment variable, with blood cell counts measured once before and twice (3 and 16 days) after treatment ended, but were also examined during 2 other experiments with blood cell counts measured once before and once after treatment. Results from all replicate experiments were in agreement with those reported here.

Statistical Analysis

Data were analyzed initially by simple descriptive statistics of mean and standard deviation using Microsoft Excel 2000 software. Minitab version 13.2 statistical software from Minitab Inc., State College, PA, was used for hypothesis testing using a 2-tailed t-test at a 95% confidence level.

Results

Body Weight Loss

Treatment-induced weight changes were monitored as a sign of general toxicity. Rats were weighed every 2 days, before, during, and after treatment (Figure 1). A dose-dependent weight loss was seen in the groups given ip treatment in that the weight of all rats given the high-dose FU+LV decreased to or just below 80% of their pretreatment weight by day 12 post treatment and they were euthanized; in contrast, the weight of those given low-dose FU+LV dipped to 90% of their pretreatment weight by day 7, and then they recovered completely. Of the groups given iv FU+LV, those given high-dose FU+LV had to be euthanized, whereas 50% of those given low-dose FU+LV did not need to be euthanized (to show this clearly the weight loss of the rats receiving the low-dose iv FU+LV, which had to be euthanized and of those that recovered are shown separately in Figure 1). From these data it can be seen that both dose and the route of FU+LV administration affected the level of toxicity. Thus, low-dose FU+LV-treated rats had greater weight loss when treatment was given iv than when it was given ip. At the high dose of FU+LV, by either route, all rats had continuous weight loss and were euthanized when their weight loss was ≥20% of their pretreatment weight.

Kinetics of Myelosuppression

To understand the dynamics of FU+LV induced myelo-suppression, rats were bled 1 day before the iv high-dose FU treatment started and 3 and 16 days after treatment ended. The results (Figure 2) show that 3 days after FU+LV treatment, there were decreases in the numbers of neutrophils, platelets, and lymphocytes and, to a lesser extent, of hematocrit, hemoglobin concentration, and red blood cells. Sixteen days after treatment, a rebound effect to greater than control levels of neutrophils and platelets was noted, while most other blood parameters had returned to normal levels by that time.

Grossly Observable Toxicities

Treated rats demonstrated several toxicities that were monitored by simple observation (Table 1). Chromodacryorrhea, an excessive secretion of fluid from the harderian gland located behind the eyeball did not occur in any of the untreated rats, saline-treated rats, or those given the ip low-dose FU+LV, but did occur in those given high-dose FU+LV (100%) by either route or iv low-dose FU+LV (83%). The severity of this toxicity ranged from slight secretion localized around the eyeball to massive secretion that completely covered the eye and stained much of the hair on the face.

Stomatitis, detected by the presence of ulcerated lesions in the mouth, is commonly seen in humans treated with FU+LV. These lesions were seen in rats that received iv low-dose or ip high-dose FU+LV (67% and 50%, respectively). Stomatitis was usually not severe, often involving only a single lesion.

Diarrhea, also a common side effect of FU in humans, was seen in those rats that received iv low-dose FU+LV or ip high-dose FU+LV (17% and 50%, respectively). Diarrhea was never very severe and usually was only detectable by discoloration of the fur in the ventral pelvic region (not shown).

Facial edema, which has not been previously documented as a toxicity of FU+LV treatment, was demonstrated by a disfiguring swelling of the face, causing a rounding of the snout, bulging over the snout, and swelling of the skin under the eyes (Figure 3). The incidence of facial edema was 100% for rats given high-dose FU+LV by either route, 83% for those given low-dose FU+LV by the iv route and 0% for those given ip. The edema varied in severity from slight broadening of the snout to massive swelling that ruptured the skin. During necropsy, incision of snouts with swelling released a clear watery fluid, indicating edema.

Enlargement of the submandibular salivary gland has also not been previously documented. This toxicity of FU+LV treatment presented itself as a hard lump on the ventral side of the throat, which could be seen in an unshaven rat (Figure 3D). Gland enlargement did not occur in the absence of other signs of toxicity. However, interestingly, the frequency of submandibular salivary gland enlargement relative to body weight loss, a general sign of toxicity, occurred according to a bell-shaped curve. Thus it was seen in (a) 83% of the rats given the ip low-dose FU+LV, which caused a low level of weight loss, (b) 100% of those given iv low-dose FU+LV, which caused an intermediate level of weight loss, and (c) 50% and 17% of those given ip and iv high-dose FU+LV, respectively, both resulting in the maximally tolerated weight loss. In a few cases the condition was seen to resolve and when it did, it took about a week.

The skin over the salivary gland was shaved at necropsy. The lump could then be clearly seen and in several cases was observed to have black areas that were later verified to be the result of necrosis (Figures 4A–4B). On 2 occasions, the submandibular salivary gland ruptured, releasing green sticky pus, indicating an infection and necrosis. The severity of this toxicity ranged from barely noticeable in the unshaven rat, to severe enlargement, resulting in immobilization of the neck. The rats with immobilization of the neck often appeared to have an arched spine (not shown). During necropsy, restricted neck movement was relieved following removal of the submandibular salivary gland that had fused to both the neck and chest. The gross appearance of normal submandibular salivary glands was that of a small pink-colored tissue, while the affected glands were large and white, some with black areas of apparent necrosis, whereas the appearance of the parotid salivary glands of treated rats were similar to that of the control rats, which was confirmed histologically (not shown).

Both normal and enlarged submandibular glands were also examined histologically (Figures 5A–5F). Histologic examination of these glands verified that they contained seromucous acini. In enlarged glands, large areas of purulent necrosis were seen, with high amounts of leukocyte infiltration involving predominately neutrophils with some lymphocytes and macrophage (Figures 5B–5D). In some areas of leukocyte infiltration, leukocytes were seen penetrating the salivary gland and nearby tissue, such as skeletal muscle, in other areas destruction of normal tissue occurred so that it was no longer recognizable (Figure 5C). Affected salivary glands contained macrophage (Figures 5E–5F). Close examination of necrotic areas and inside certain macrophages show the presence of both free and phagocytized bacteria, respectively (Figures 5E–5F).

Discussion

These studies were undertaken to define baseline parameters prior to undertaking the evaluation of the possible role of the immune system in the IL-15-mediated potentiation of the antitumor efficacy of FU+LV combination treatment reported by Cao and Rustum (1998). The purpose was to verify the toxicities reported for the FU+LV combination when administered at the doses and by the routes that had been used in their study. The toxicities reported by Cao and Rustum (1998), and by many others (Haines et al., 1967; Radley et al., 1980; Speyer et al, 1980; Gyves, 1985; Donowitz and Quesenberry, 1986; Campora, 1987; Bruckner, 1988; Mahteme et al., 1998; Regelson and Parker, 1986; Sugarbaker et al., 1985), namely: dose-dependent weight loss, depression of hematological parameters followed by rebound, chromodacryorrhea, stomatitis, and diarrhea were confirmed. In addition facial edema and enlargement of the submandibular salivary gland was observed.

To our knowledge, the facial edema and enlargement of the submandibular salivary gland induced by FU+LV have not been reported previously. Facial edema incidence correlated with the degree of body weight loss, occurring in all rats that suffered body weight loss severe enough to require euthanasia and with decreasing frequency in rats with lower overall weight loss. Interestingly, in our studies, the incidence of facial edema was higher than that of either stomatitis or diarrhea, the dose-limiting toxicities. Edema may have contributed to the enlargement of the submandibular salivary gland, but the cause of edema is not known.

Enlargement of the submandibular salivary gland was never seen in the absence of other toxicities but its incidence, relative to body weight loss, was described by a bell-shaped curve. Thus, the treatments causing the greatest body weight loss did not cause the greatest incidence of enlarged salivary glands. Histologic examination of the enlarged glands indicated that a bacterial infection had occurred as well as an inflammatory reaction and that the salivary gland parenchyma was destroyed. Since rats housed separately still presented with enlarged salivary glands, the possibility that bacteria were introduced due to skin lesions as a result of biting was excluded. The vendor’s health report and the routine sentinel animal-based health screening of the animal facility ruled out that the infection was pre-existing. This suggests that the bacteria were opportunistic pathogens.

It has been shown by others that FU is present in salivary secretions and can damage glands and decrease saliva production (Celio et al., 1983; Abbas et al., 1986; Hayashi and Watanabe, 1990; Slavik et al., 1993; Joulia et al., 1999). Loss of saliva production can increase the susceptibility to oral and salivary gland infections. Direct damage to the mucosa weakens the integrity of the tissue and when this is coupled with blockage of the salivary gland, or decreased saliva production and immunosuppression, bacteria may enter the interstitium (Sonis et al., 1990, 1992). Interestingly, blockage of the salivary glands can occur from edema, resulting in bacterial infection and inflammation, i.e., sialoadenitis (Gnepp and El-Mofty, 1996). This suggests a possible link between the facial edema and the enlarged submandibular salivary gland. However, these 2 toxicities probably had different etiologies. Facial edema (17 cases) was almost exclusively seen in those treated rats that had body weight loss severe enough to necessitate euthanasia (15 out of 17 euthanized), while submandibular salivary gland enlargement (15 cases) was seen equally in those that were euthanized (7 cases) and those that survived treatment (8 cases).

The nonlinear relationship between the severity of submandibular salivary gland enlargement and the overall sign of toxicity, i.e., body weight loss, suggests a complicated etiology. If the cause of submandibular salivary gland enlargement was simply opportunistic bacterial expansion subsequent to immunosuppression, then a linear and not a bell-shaped relationship would have been expected. It may be suggested that this relationship can be explained if the salivary gland enlargement is related to the ability to carry out an inflammatory response. Thus, it can be proposed that an inflammatory response was not triggered when overall toxicity was below a certain level or when toxicity was so severe that the ability to generate an inflammatory response was lost.

An immunologically depressed state may have occurred just after treatment with iv high-dose FU+LV, since neutropenia was noted 1 (not shown) to 3 days posttreatment.

Neutropenia was followed by a rebound effect (measured 16 days posttreatment) in which leukocytes may have started to increase at the time the submandibular salivary gland became enlarged (first detected 5 days after treatment finished). An association between the presence of bacteria and the condition was supported by the observation that only the submandibular gland was affected and the other salivary glands had neither noticeable bacterial infections nor enlargement. In fact, one of the most interesting aspects of this toxicity is that it occurred consistently in the submandibular salivary gland, not in the parotid salivary gland, and was never detected in the sublingual salivary gland.

In the rat, the sublingual gland lies at the anterolateral surface of the submandibular gland. Therefore, because of the severity of the submandibular salivary gland enlargement, a similar enlargement of the sublingual gland should have been detected, as the 2 glands are within the same fibrous capsule. The most probable cause that can be suggested based on the current data, is that the FU-induced temporary systemic immunosuppression together with possible FU-induced epithelial damage in the salivary gland and decreased saliva production allowing for opportunistic bacteria (e.g., oral resident bacteria) to set up a colony in the stroma of the gland. The presence of these bacteria would then have initiated an inflammatory response as immunocompetence rebounded. The link between chemotherapeutic agent-induced immunosuspression and increased risk of infection is well known (Rolston and Bodey, 1997). However, the reason why this effect occurred only in the submandibular salivary gland and not in the other salivary glands has not been determined and would require further investigation.