Mesovarian leiomyomas in the rat.

Prolonged treatment with two chemically distinct beta-stimulants, Salbutamol and Terbutaline, resulted in mesovarian leiomyomas in Sprague-Dawley rats. Development of these tumors induced by Salbutamol was prevented by concurrent administration of the beta-blocker Propranolol. Mesovarian leiomyomas induced by Salbutomol did not show any regression or progression during a 44-week postdosing recovery period. This report also gives the first recorded incidence of spontaneous mesovarian leiomyomas in the rat.


Introduction
Primary leiomyomas of the ovaries are uncommon in man; one review recorded only five cases from a collection over a 36-year period (1). Smooth muscle cell tumors of the ovaries are likewise rare in all species of animals. We found no reported cases of spontaneous mesovarian leiomyomas in rats in the published literature. In contrast to spontaneously occurring smooth muscle cell tumors, mesovarian leiomyomas can be induced in rats by prolonged administration of ,8-stimulants.
This report describes the pathologic features and compares the incidence of mesovarian leiomyomas induced in female rats by two ,B-stimulants, Salbutamol sulfate and Terbutaline sulfate, and shows the effects on the incidence of Salbutamol-induced leiomyomas of concurrent administration of the 13-blocker, Propranolol. In addition, results from an extensive review of ovaries from untreated rats are presented.

Materials and Methods
The test animals used were female Charles River CD (Sprague-Dawley derived) rats, and the compounds were administered orally in the diet. The study was divided into two parts. Experimental design for Part I is given in Table 1. Part II of the study consisted of 200 female rats that were given Salbutamol at a dose level of 20 mg/kg/day for 80 weeks. Fifty rats were killed at week 80, and the remaining survivors (98 rats) were held without treatment for a postdosing recovery period up to week 124. At this time all survivors were killed. All animals were given a complete postmortem examination and tissues were fixed in 10% neutral buffered formalin. Only the results ofthe mesovarian leiomyomas are included in this report. In addition, a retrospective review of ovary tissue diagnoses from 7748 untreated rats in the Huntingdon Research Centre database was *Huntingdon Research Centre, Huntingdon, Cambridgeshire, PE18, 6ES, England.  CThe dose of Terbutaline was doubled from week 63 as it was found to give less than intended plasma concentrations in the treated rats (2). dThe dose of the Propranolol was that found to effectively abolish the increase in heart rate caused by oral Salbutamol in the rat (2). conducted to establish the spontaneous incidence of mesovarian leiomyomas.

Results
Part I A summary of the mesovarian smooth muscle lesions observed in Part I of this study is given in Table 2. Both ,B-stimulants, Salbutamol and Terbutaline (groups 3, 4, and 5), produced mesovarian leiomyomas. There were no rats with leiomyomas among the controls nor in the group given Salbutamol plus Propranolol.
Eighteen of the rats with leiomyomas had grossly detected lesions. Nine of these lesions were located on the right side, six were bilateral, and three were seen on the left side. These tumors varied from 1 mm to 20 Forty-one ofthe 200 rats had macroscopic mesovarian mm in diameter. The earliest appearance ofa leiomyoma lesions present as swellings, nodules, or masses. in this part of the study was at week 56 in a decedent Twenty-nine of these grossly detected lesions were on rat from the group receiving Salbutamol at 20 mg/kg/ the right side, nine were bilateral, and three were on day.
the left side. The earliest appearance of a leiomyoma in this part of the study was at week 63. The lesions varied Part II from 2 mm in diameter to a 29 x 24 x 16 mm irregular mass, the largest tumor. The lesions were located most The incidence of leiomyomas seen in Part II of this frequently in the mesovarium extending into the hilar study is given in chronological order in Table 3. There region of the ovary, dorsal to the ovary (Plate 1), and were 52 intercurrent deaths during the dosing period sometimes freely in the mesovarian ligament, up to 12 (up to 80 weeks), and 50 more rats were killed at 80 mm away from the tip of the uterine horn. Macroscopic weeks. Among these 102 rats, there were 17 rats with lesions appeared as fusiform swellings, spherical or mesovarian leiomyomas. During the postdosing recov-ovoid nodules, or as irregular bosselated masses (Plate ery period, there were another 22 rats with mesovarian 2). Cut surfaces revealed dense whitish tissue with a leiomyomas (among the remaining 98), including one faintly whorled pattern (Plate 3). Lesions were wellwhich was killed at week 124. circumscribed, but had no distinct encapsulation.

Histological Features
Histologically, leiomyomas appeared as well-circumscribed growths of smooth muscle fibers (Plates 4 and 5). The muscle fibers appeared as interlacing bundles, with a crisscrossing pattern and were sometimes arranged in whorls (Plate 6). The muscle cells were long, with abundant eosinophilic cytoplasm and cylindrical, cigar-shaped nuclei with blunt ends (Plate 7). In some of the microscopic fields, the nuclei appeared ovoid or circular depending on the sectioning angle on the bundle of muscle fibers. The tumor cells stained yellow with van Gieson and red with Mason's trichrome. Sparse interbundle collagen was also present in varying proportions.
Irregular areas of smooth muscle tissue in excess of the usual amount found in the mesovarium, the suspensory ligaments, or in the ovarian hilus were diagnosed as smooth muscle hyperplasia. In these instances, although the smooth muscle tissue was focally in excess, the lesions lacked the characteristic patterns of interlacing bundles or whorls seen in the tumors and appeared as areas of irregular or parallel smooth muscle fibers (Plate 8).
The review of the ovaries carried out to establish the occurrence of mesovarian leiomyomas among untreated rats revealed four cases in 7748 rat ovaries examined. These four cases had morphological features identical to those described for the induced tumors described previously.

Discussion
Leiomyomas recorded in this study arose from the smooth muscle normally present in the mesovarium. This muscle tissue extends into the ovarian hilus and anteriorly into the ligaments. None of the tumors examined showed any evidence of malignancy. The tumors tended to occur more frequently on the right side than the left, which was similar to a previous observation (3).
Mesovarian leiomyomas were induced in rats by the administration of two chemically distinct adrenergic stimulants, Salbutamol and Terbutaline. Other ,-stimulants like Soterenol hydrochloride and Mesuprine hydrochloride are known to induce these tumors in the rat (3,4). Two other agonists, Zinterol and Reproterol, were also reported to have induced leiomyomas in the rat (2). A 12-month study in rats with high doses of Mabuterol revealed 3 out of 56 rats with mesovarian leiomyomas (5).
The induction of mesovarian leiomyomas in rats appears to be a general feature of P-stimulants. That the induction of these benign tumors is a function of adre-nergic stimulation was further evidenced by the fact that the concurrent administration of the adrenergic blocker, Propranolol, prevented their development. Mesovarian smooth muscle in the rat is known to contain P2-adrenoceptors, and it is postulated that prolonged and intense activation of ,32-adrenoceptors that mediate cellular relaxation might give rise to leiomyomas (6).
In Part II of this study, female rats treated with Salbutamol 20 mg/kg/day for 80 weeks followed by a postdosing recovery period of 44 weeks had a similar incidence of mesovarian leiomyomas during the treatment and recovery periods. Since these tumors were still detected toward the end of the recovery period, and there were no appreciable differences in the size of the masses detected, it can be concluded that these lesions were neither reversible nor progressive.
Beta-stimulants are known to induce mesovarian leiomyomas in different strains of rats such as Sprague-Dawley derived (Charles River CD strain), pigmented Long Evans, and Wistar derived (Jcl) rats (2,3,5). 13stimulants are not known to produce mesovarian leiomyomas in mice or other species of laboratory animals. This class of pharmacological compounds has been extensively used for more than 15 years in man, and there is no evidence of any increased incidence of smooth muscle tumors among users ofthese drugs (7). It is apparent from this report that these tumors in the rat are not unique to 1-stimulants but do occur spontaneously, albeit exceptionally rarely.