EFFECT OF BETA-AMINOPROPIONITRILE ON T U M O R METASTASES IN T H E R A T

MARK GORDON, MD, STUART ZYKORIE, BA, BERNARD GARDNER, MD,* JOSEPH PATTI, BS, AND HENRY GRAY, MS

Rats treated with either beta-aminoproprionitrile or saline received intramesen- teric venous Walker sarcoma. Results showed no differences in hepatic takes but a significant increase in pulmonary metastases in BAPN-treated animals. Bone collagenase levels were the same in both groups. Histologic studies revealed a disruptive influence of BAPN on collagen integrity of large vessels. The results imply that the lung is more sensitive to this effect than liver.

OTENTIATION OF METASTASES OF WALKER SAR- P coma by parathyroid extract and Yoshida sarcoma by parathyroid hormone has been previously demonstrated.7Jl Histologic studies of tumors from those experiments showed a decreased fibrous reaction which may have been secondary to a depression of the host's response to the tumor, increased collagenoly- sis, or decreased polymerization of ground substance.2.7 The results could not have been due to alteration of serum calcium levels since such changes do not influence tumor metas- tases of Walker sercoma.8 In the following study, treatment with beta-aminopropionitrile (BAPN), which produces the syndrome of osteolathyrism in rats through defective col- lagen formation,14.16 was investigated to see if collagen integrity plays a critically defensive role against the spread of malignant tumors.

MATERIALS AND METHODS

One hundred male Sprague-Dawley rats were divided into equal groups of either 100 or 250 g. Half of each group received saline or 100 mg of beta-aminopropionitrile (BAPN) daily, intraperitoneally for 11 days. On the fifth day, laparotomy was performed under ether anesthesia. Intramesenteric venous injec- tions of 500,000 Walker sarcoma cells in the 100-g rats and 1,000,000 cells in the 250-g rats were ma&. After the 11 th day, treatment was continued three times weekly until sacrifice at

From the Department of Surgery, Downstate Medi- cal Center, State University of New York, 450 Clarkson Ave., Brooklyn, N.Y.

Supported, in part, by Grant CA-10084, U.S.P.H.S. John and Mary R. Markle Scholar in Academic

Medicine. Received for publication April 28, 1971.

8 weeks. Exploratory laparotomies were per- formed 3 weeks after tumor inoculation to de- fine early hepatic takes, and animals dying prior to this were excluded from the results. Histologic examination of mesenteric lymph nodes, spleen, liver, lungs, kidney, and bone was performed. Bone collagenase activity was measured in femurs from freshly sacrificed an- imals by a technique involving the release of C 14 from a labelled collagen gel.4

RE~ULTS

At laparotomy, 6 animals in each group had liver metastases. Sixty-five animals survived, 30 of whom received the smaller tumor dose (14 BAPN treated and 16 controls). Of the re- maining 35 animals, there were 19 BAPN treated and 16 controls. The combined results demonstrate 45% hepatic takes in animals treated with BAPN compared to 41% in the controls (Table 1). However, in the BAPN- treated animals, 14/33 developed pulmonary metastases compared to 3j32 in the control group (p < ,005).

Measurements of collagenase (Table 2) show no significant difference between the control and treated groups. Animals pre- treated with parathyroid extract (PTE) are in- cluded for comparison and have significantly increased collagenase levels. Examination of H and E-stained sections of bone demon- strated no metastases in either group. The effect of BAPN on the treated group was evi- denced by medial edema of the aorta and frag- mentation of elastic fibers (Fig. l), which was not seen in saline-treated rats (Fig. 2). Early disruption of the arterial wall was noted in several animals (Fig. 3). No morphological

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510 CANCER Febrtiaiy 1972 Vol. 29

TABLE 1. Incidence of Hepatic and Pulmonary TABLE 2. Collagenase Levels in Femora of Rats Metastases in Rats Treated with Beta

Aminopropionitrile (BAPN) Treated with Parathyroid Extract (PTE)*

or Beta Aminoproprionitrile (B.L\PN)

Collagenolytic activity+ ~ _ _

Liver Lung (Tumors/tot:d (Ttimors/total Normal bone 3 . 3 %

aniinalz) animals) PTE-treated bone 8 1% Saline control bone 2.5% & . 1.5 (S.E.)

BAPN 15/33 (45%) 14/33* (42%) Br\PN-treated bone 2 8% 29 (S.E.) Saline 1 3 / 3 2 (41%) 3/32* (9%) *Generously supplied by Dr. .\. S. Ridolfo, Eli

Lilly & Co. * p < 0.005. t Per cent release of CI4 label per 100 mg tissue.4

No. 2 BETA-AMINOPROPIONITRILE AND TUMOR METASTASES - Gordon et al. 511

FIG. 3 ( top) . High-power view of aorta of BAPN-treated rat showing disruption of the media and disorganization of the elastic fibers (~325). FIG. 4 (bot tom). Pulmonary parenchyma in a BAPN nontumor-bearing rat. The septae appear normal (xl10).

differences attributable to BAPN were found on elastic van Gieson’s stain of BAPN-treated normal or tumor-bearing pulmonary paren-

b r a t demonstrating disorganization of the medial elastic fibers (~110). FIG. 2 (bottom). Saline-treated control. Note the regular arrangement of elastic fibers compared to Fig. 1 (x110).

chyma (Figs. 4, 5, 7). Tumor involvement was usually diffuse (Fig. 6), and there was no vas- culi tis, thrombus formation, or endothelial al-

512 CANCER February 1972 Vol. 29

teration. Examination of lymph nodes, spleen, liver, and kidney failed to show any apprecia- ble differences between saline and BAPN- treated animals.

pea, Lathyrus latifolius, that produce neuro- logic (neurolathyrism) and connective tissue (osteolathyrism) disorders in man. BAPN pro- duces a relatively pure state of osteolathyrism, with changes in skin, dental tissues, blood ves- sels, tendons, and bone. Lathyrism in labora- tory animals gives a true experimental colla- gen disease for study. T h e defect consists of weakening and/or absence of intramolecular

DISCUSSION

Beta-aminopropionitrile is one of several naturally occurring substances from the sweet

No. 2 BETA-AMINOPROPIONITRILE AND TUMOR METASTASES Gordon et al. 513

FIG. 7 (top). High-power view of a BAPN-treated rat with pulmonary metastases (~325). FIG. 8 (bottom). Hepatic metastases in a BAPN-treated rat. Note the tumor cells are deeply stained and small with a dense fibrous stroma (~110) .

cant fibrous reaction ( ~ 1 1 0 ) . FIG. 6 (bottom). Pulmonary metastases in a BAPN-treated rat demonstrating the diffuse nature of the metastases with minimal fibrous reaction (X 110).

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FIG. 9 (top). Hepatic metastases in a saline control rat. The morphology is similar to that seen in the BAPN-treated animals (X110). FIG. 10 (bottom). Hepatic metastases in a PTE-treated animal. Note that the cells are plump and less pyknotic. The fibrous response is minimal com- pared to Fig. 9 (xll0).

515 No. 2 BETA-AMINOPROPIONITRILE AND TUMOR METASTASES - Gordon et al.

and intermolecular cross linkages in collagen aggregates. Although the exact mechanism of action of lathyrogenic agents is unknown and has been the subject of intense investigation, they probably act by blocking carbonyl groups on the collagen molecule, and by inhibiting reactions involved in the oxidation of lysine to aldehydes.14J6 Other prominent theories of action concern proteolytic enzyme release, and inhibition of cellular respiration.15 Associated defects in connective tissue have been found, but whether these are primary or secondary is unclear. Other measurements of collagenolytic activity in bone indicate that collagenase is not the mediator in this disorder.12.14

Research into the nature of tumor metas- tases has shown several distinct events to be necessary: 1. embolization; 2. tumor cell ag- glomeration with capillary plugging or ves- sel wall attachment; 3. fibrin deposition and early clot formation, and 4. invasion through the vessel wall into the tissue. Gardner et al.,a investigating the effects of altered connective tissue states and calcium metabolism, in an at- tempt to reproduce the work of Pentimalli,l3 failed to induce skeletal metastases with either exogenous vitamin D or parathyroid extract.4 Jones8 was unable to show a change in distant metastasis with exogenous vitamin D. Re- cently, Jones and Gardner? showed that intra- portal tumor injection in normal or thyropar- athyroidectomized rats treated with high doses of parathyroid extract yielded a significant in- crease of distant pulmonary metastases com- pared to control rats. In this study, the colla- genase activity was markedly elevated. They postulated that interference with the immune response or alterations in the ground sub- stance of the tissue were possible mechanisms for this result. There is no evidence that BAPN affects host resistance. In fact, two short-term clinical trials of BAPN in patients with sclerodermao or undergoing tendon repair12 have shown rapid development of al- lergic phenomena.

&man et aL,l investigating hyaluronidase, could not demonstrate an influence on tumor

spread. They postulated that they may have been using an inappropriate tumor system, an- tihyaluronidase may have been formed, or that hyaluronidase does not promote metas- tasis. They investigated subcutaneous growths which may not have evaluated the effect on those stages occurring after embolization. Wood17 demonstrated increased metastases after pretreatment with hydrocortisone, growth hormone, cold and formalin stress.

In the only other study of tumor behavior in BAPN-treated rats, McCrary et a1.10 noted no increase in the rate of growth or frequency of takes in subcutaneous tumor injections. They did note increased local bone invasive- ness, but attributed this to the increased po- rosity of lathyritic bone.

Our current results demonstrate that tumor metastases in liver are unaffected by BAPN injections. The fibrous reaction to metastatic deposits is similar to that seen in saline- treated animals (Figs. 8, 9). In our previous study demonstrating increased liver takes with PTE, the fibrous reaction was less evident (Fig. lo). Therefore, it appears that lung reacts differently to BAPN than does liver ac- counting for the diffuse pulmonary infiltra- tion of tumor in the absence of the kind of fib- rous response seen in hepatic sections. Light microscopy, however, failed to reveal signifi- cant differences in stromal architecture which could account for this. Microscopic changes in the aortas of the BAPN-treated animals indi- cate that the drug was effective in producing disturbances in collagen metabolism.

The effectiveness of BAPN in producing more pulmonary metastases could be due to the promotion of increased numbers of tumor cells into the hepatic venous circulation or a selective influence on pulmonary collagen me- tabolism. Increased numbers of tumor cells passing the hepatic capillary filter should have been associated with a decreased number of hepatic takes, but this was not observed. I t ap- pears likely, therefore, that sensitivity of the lung to the effects of BAPN accounted for the observed results.

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Failure of osteolytic agents to induce skeletal metastases in rats with sarcoma. Cancer 18:1085, 1965.

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