Cytotoxicity of cisplatin in rat intestine

TOXICOLOGY AND APPLIED PHARMACOLOGY 60, 354-359 (1981)

Cytotoxicity D. CHOIE,

DAVID Laboratory

DANIEL

of Cisplatin in Rat intestine S. LONGNECKER,’

AND MARION

P. COPLEY

of Chemical Pharmacology. Building 37, Room 5A13, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205

Received January 28, 1981; accepted March 26, 1981 Cytotoxicity of Cisplatin in Rat Intestine. CHOIE, D. D., LONGNECKER, D. S., ANDCOPLEY, M. P. (1981). Toxicol. Appl. Pharmacol. 60, 354-359. Therapeutic treatment with cisplatin (cis-dichlorodiammineplatinum-II), an antitumor agent, produces toxic side effects involving kidney, GI tract, and bone marrow. Intestinal cytotoxicity of cisplatin was characterized in rats following a single ip dose (5 mg/kg). Cellular necrosis and inhibition of mitosis in the intestinal epithelium were maximal on Days l-2 and were most severe in the ileum, but mucosal lesions were recovered by 5-7 days. Crypt and villus cell populations were reduced most in the ileum (60-70%). followed by the jejunum (45-60%) and the duodenum (35-40%). Stomach and colon had few mucosal lesions. GI tissues assayed for platinum concentrations indicated no preferential localization of cisplatin in any segment of small intestine. Histologic evidence suggested that proliferating epithelial cells in the crypt are the major targets for cisplatin cytotoxicity.

Cisplatin (cis-dichlorodiammineplatinum-II) is an inorganic coordination compound that has antitumor activity against a number of animal tumors (Rosenberg, 1977; Prestayko et al., 1979) and human neoplasms, notably testicular, ovarian, and epidermoid carcinomas (Hill et al,, 1975; Rozencweig et al., 1977; Burchenal, 1978; Sako et al., 1978). The major toxic side effects of cisplatin involve the kidney, the gastrointestinal (GI) tract, and the bone marrow. Renal toxicity is the most serious dose-limiting factor (Talley et al., 1973; Madias and Harrington, 1978; Von Hoff et al., 1979). However, cisplatin nephrotoxicity can be ameliorated clinically by prehydration and osmotic diuresis (Hayes et al., 1977; Frick et al., 1979). GI toxicity, on the other hand, remains troublesome, causing clinical symptoms in cisplatin treatment (Talley et al.,

1973; Hill et al., 1975; Von Hoff et al., 1979). So far, little work has been done on cisplatin cytotoxicity in the intestine and on the mechanisms of nausea and vomiting. In the present study we have analyzed qualitative and quantitative parameters of cytotoxicity of cisplatin in GI mucosa of rats. Our results show that a single dose of cisplatin produces acute transient epithelial necrosis among dividing cells in the crypts. The lesion is most severe in the ileum on Days 1 and 2. METHODS Male Fischer 344 rats (170-210 g) from Charles River Breeding Labs (Wilmington, Mass.) were maintained on Purina Laboratory chow and tap water at all times. Cisplatin was provided by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, NCI, and dissolved in physiological saline (I mg/ml) within one hour before use. Groups of four rats were injected ip with a single dose of cisplatin (5 mg/kg) between 10 AM and 2 PM, and

’ Present address: Department of Pathology, Dartmouth Medical School, Hanover, N.H. 03755 0041-008X/81/1

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were sacrificed at comparable times on Days 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, and 28. Controls were injected similarly with physiological saline, and sacrificed in groups of two rats. In each animal, two pieces of tissue were taken from stomach, duodenum, jejunum, ileum, cecum, and colon. One set of the tissues was prepared for histology (5-p section) and stained with hematoxylin and eosin. Tissues of the other set were rinsed in saline to remove the intestinal content, digested in nitric acid, and assayed for platinum concentrations by flameless atomic absorption spectrometry (Perkin-Elmer Model 403) at 266 nm (Litterst et al., 1976; Choie et nl., 1980). To estimate changes in cell populations in small intestinal mucosa, epithelial cells of crypts and villi were counted in duodenum, jejunum, and ileum following treatment with cisplatin. In each segment of small intestine, at least five longitudinal sections of villi and of crypts, respectively, were scored for total number of epithelial cells and mitotic figures. Statistical significance was evaluated using the Student t test.

RESULTS Following treatment with cisplatin, animals had diarrhea on Days l-3, and the gastrointestinal tissue retained a relatively high concentration of cisplatin. Histologically, acute necrotic enteritis was seen in the mucosa of the small intestine. The primary targets for cisplatin cytotoxicity were dividing cells in the crypts. The intestinal mucosa was restored to near control state by Day 5. Comparatively minimal injuries were observed in the stomach, cecum and colon. Differential Cytotoxicity of Cisplatin in Intestinal, Mucosa Duodenum One of the early lesions was focal necrosis in crypt epithelium. Dne day after treatment with cisplatin, a few necrotic cells were seen in less than 10% of the duodenal crypts (Fig. 1A). Mitotic figures were reduced to about 50% of controls on Days 1 and 2, but rapidly recovered by Day 3, and increased above controls by 50% on Day 4 (p < 0.05). Although there was some shortening of villi,

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the general architecture was maintained.

355 of villi and crypts

Jejunum On Day 1, necrotic cells were found in about 30% of the jejunal crypts. Necrotic cells were usually located in the midregion of the crypt epithelium. Mitotic figures were reduced by two-thirds. The inhibition of mitosis apparently slowed migration of epithelial cells from the crypt to villus, resulting in flattening of the remaining cells to a cuboidal or squamous configuration to maintain an epithelial lining. Many crypts were atrophied on Day 2, villi were shortened in height, and lacteals were’ dilated (Fig. 1B). Mitotic activity began to increase on Day 3, and the epithelial lining of the crypts and villi was restored to near control state by Day 5.

Ileum In the entire GI tract, ileal mucosa was most severely damaged following cisplatin treatment. About 50% of ileal crypts contained one or more necrotic cells-on Day 1. Mitotic figures were rare. On Days 2 and 3, the surviving epithelial lining cells were flattened, and villi were shortened, often to about half their normal height (Fig. 1C). Some villi were blunted to stubby structures partially fusing with neighboring villi in the base region. Almost all ileal crypts were atrophic, and many of them appeared to be separated from villus epithelium, forming round enclosures like cysts. These cryptic cysts were usually made of lo-20 cells in longitudinal section containing a small group of Paneth cells at the basal regions. Occasionally, inflammatory cells were seen, and crypt abscesses containing polymorphonuclear leukocytes and cell debris were also observed in some crypts. On Days 3-5, there were signs of active regeneration of epithe-

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AND COPLEY

FIG. 1. (A) Photomicrograph of duodenal crypts after a single ip injection of cisplatin (5 mg/kg) in rat, showing necrotic cells (arrow) in the crypt epithelium. H&E staining (X260). (B) The jejunal crypts 2 days after a dose of cisplatin (5 mg/kg) in rat. The crypts are atrophied, necrotic cells are seen, and no mitotic figures are found in the crypts. Epithelial lining cells are flattened (arrow). H&E staining (X260). (C) The ileal mucosa 2 days after a dose of cisplatin (5 mg/kg) in rat. The crypts and villi are markedly atrophic. Villi are shortened and partially fused in the basal region. The lacteals are dilated (L). H&E staining (X140).

lium. Mitotic activity was increased, crypts and villi were larger, and the cystic crypts were elongated in height eventually reconnecting with the villus epithelium. During this period atrophic crypts were admixed with hyperplastic crypts. The regenerating epithelial cells had enlarged nuclei, which were often irregular in shape with some margination of chromatin, prominent nucleoli, and basophilic cytoplasm. Many villus epithelial cells showed irregular shape and cytoplasmic blebbing.

tion was significantly higher in the cecum than in other parts of the GI tissue on Day 1 (p < 0.05). On the fourth day after a single 2l-

Stomach IA.,,-..

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To estimate cisplatin concentration in the tissue, platinum was assayed in segments of GI tissues (Fig. 2). There was no preferential accumulation of cisplatin in any segment of the small intestine, but platinum concentra-

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4

5

6

7

DAYS

FIG. 2. Tissue concentration of platinum in various segments of the GI tract over a period of 7 days following an ip injection of cisplatin (5 mg/kg) in rats on day 0. Platinum was assayed for acid-digests of tissues by flameless atomic adsorption spectrometry.

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dose of cisplatin, platinum was barely detectable in any part of the GI tract. Changes in Cell Population Mucosa

in Intestinal

In normal animals, the height of the villi becomes gradually shorter from the duodenum to the ileum, whereas crypt size is comparable throughout the small intestine. In the duodenum, treatment with cisplatin reduced the epithelial cell population in crypts by 35% on Day 2, resulting in 40% reduction in villus cell population on Day 3 (p < 0.05) (Fig. 3a). There was a compensatory hyperplasia in the crypts on Day 4, which resulted in an increase in villus cell population on Day 5. In the jejunum, cisplatin reduced crypt cell populations by 45% on Day 2, and reduced villus cell populations by 60% on Day 3 (p < 0.05) (Fig. 3b). Regeneration of epithelial lining cells was somewhat slower in jejunum than in duodenum, as determined by peak hypertrophy of jejunal crypts, which occurred on Day 5. The evaluation of cell populations in the ileum was complicated because many villi were blunted and fused with each other and because many crypts were atrophied to residual cysts. In those crypts and villi still

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357

maintaining anatomical resemblance to normal epidhelium, the cell population was reduced by nearly 60% in crypts on Day 2 and by 70% in villi (p < 0.05) (Fig. 3~). It should be noted that the cystic crypts usually contained fewer cells per section than open crypts. Therefore, if cystic crypts were taken into consideration, the cell populations in ileal crypts would be smaller than estimated.

DISCUSSION It is thought that at the cellular level, DNA is the primary target of cisplatin (Harder and Rosenberg, 1970, Pascoe and Roberts, 1974; Rosenberg, 1977). Cisplatin interacts with DNA producing interstrand crosslinking, DNA-protein crosslinks, and inhibition of DNA synthesis (Heiner and Bassleer, 1976; Bergerat et al., 1979; Zwelling et al. 1979). Cisplatin resembles the bifunctional alkylating agents in its mode of action (Rosenberg, 1977). However, a recent study has indicated that significant fractions of intracellular cisplatin were localized in microsomes, mitochondria, and plasma membranes, as well as in the nuclei (Choie et al., 1980). Thus, it remains to be resolved to what extent the reaction of cisplatin with DNA is responsible for cytotoxicity.

1

FIG. 3. (a) Changes in duodenal cell populations in crypt and villus epithelia following an ip dose of cisplatm (5 mg/kg) in rats. Controls are shown on Day 0. (b) Cytotoxic effect of cisplatin (5 mg/kg) on jejunal cell populations in rats. Controls are shown on Day 0. (c) Changes in heal cell populations in crypt and villus epithelia in rats following a single administration of cisplatin (5 mg/kg). Controls are shown on Day 0.

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In the present study, the target cells of cisplatin cytotoxicity were located predominantly in the mid region of crypts of the small intestine, as determined by the appearance of cell necrosis. Since mitotic figures were most frequently found in the midregions of crypt epithelium, the results indicate that proliferating cells were the primary target of cell killing by cisplatin. Cytokinetic studies also support the view that dividing cells are more susceptible to cytotoxicity of cisplatin than nondividing cells. It has been shown that cisplatin irreversibly blocks in animals and man proliferating cells in S and G2 phases (Heiner and Bassleer, 1976; Bergerat et al., 1979). Similarly, several antitumor agents, such as adriamycin, methotrexate, and cyclophosphamide, inhibit mitosis in murine small intestine and preferentially kill cells in S phase (Sobhon et al., 1977; Jeynes and Altman, 1978; Dethlefsen and Riley, 1979). In our study cisplatin-induced inhibition of mitosis was only temporary, lasting 1-2 days. By Day 3, there was an active regenerative cell proliferation in crypts throughout the small intestinal mucosa with subsequent normal cell migrations toward the tips of villi. This suggests that those cells in the crypts with proliferative potential were largely unaffected by a dose of cisplatin that was cytotoxic to dividing cells. Maximal cell necrosis seen on Days 1 and 2 seemed to be related to the relatively high tissue accumulations of cisplatin in the intestine during the first 3 days. Our preliminary studies also indicated that intestinal cytotoxicity of cisplatin was dose dependent. At a high dose of cisplatin (6 or 7 mg/kg), intestinal lesions were quantitatively more severe, but the course of regeneration of mucosal epithelium was similar to that seen at a low dose (not shown). In the GI mucosa, the effect of cisplatin cytotoxicity was probably modified by a number of unknown factors, such as pH, because stomach, cecum, and colon were spared from intense cell necrosis, while tissue concentrations of cispla-

AND

COPLEY

tin were comparable in all segments of the GI tract. It may be pointed out that the maximum tissue concentration of cisplatin in the intestine was at least lo- to 20-fold less than that found in the kidney. (Litterst et al., 1976; Choie et al., 1980). The renal toxicity was thought to be related to this high accumulation of cisplatin in the kidney (Madias and Harrington 1978; Choie et al., 1980). Histopathologic changes and reductions in cell populations suggest that cisplatin cytotoxicity was most severe in the ileal mucosa. Since there was no significant differential distribution of cisplatin in segments of small intestinal tissues, the pronounced injury to ileal mucosa might be due to an intrinsic susceptibility of ileal crypt cells to cisplatin. Another possibility is that small amounts of cisplatin or its metabolites could be excreted into the intestinal lumen, and that cisplatin in the cells sloughed from the proximal portion of the small intestine, could have become a secondary source of exposure for the ileal mucosa. In such a case, the actual amount of cisplatin taken up by the ileal cells may be too small to be detected by the analytical method used, but significant enough to cause cytotoxicity by a direct interaction at the cellular level. What seems to be certain is that there were no qualitative distinctions in the types of lesions seen in the ileum compared to those in other segments of intestine, although cystic crypts were found mostly in the ileum. It is informative that inhibition of cell proliferation in intestinal mucosa following a single treatment of cisplatin is reversible within 3 days, and that the damge is dose dependent. The exact mechanism of intestinal cytotoxicity of cisplatin is not clear. However, available evidence suggests that a direct reaction of cisplatin with DNA with the resulting disruption of DNA metabolism or function could be the major factor in cytotoxicity (Pascoe and Roberts, 1974; Rosenberg, 1977; Zwelling et al., 1979). In addition the present data suggest that the

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cytotoxicity is probably linked with the proliferative activity of epithelial cells, presumably associated with a specific phase of the cell cycle. Unfortunately, the present animal experimental system does not allow one to determine whether or not the intestinal lesions are causally realted to clinical symptoms, such as vomiting, because murine animals do not have such reflexes. Further studies are needed to clarify the cellular and molecular basis of cisplatin cytotoxicity, and the cause of the clinical symptoms of the GI toxicity, which might lead to the ultimate control of the toxic side effects in the treatment of cancer patients with cisplatin. ACKNOWLEDGMENT We thank Dorothy J. Powell for her excellent technical assistance.

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