Development of a system for controlled release of benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, and phorbol ester for tumor induction in heterotopic tracheal grafts.

The utility of the trachea! transplant model as a tool in in vivo carcinogenesis studies depends largely on the development of a good drug delivery system affording reproducible, sustained carcinogen release. Previously used methods have proven to be less than satisfactory. We studied the release of benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, and 12-O-tetradecanoylphorbol-13acetate from pellets of varying composition. In vitro release of the two polycyclic hydrocarbons (PCH) from beeswax pellets showed little variability, regardless of PCH concentration. In marked contrast, in vivo release was highly variable, particularly at high PCH concentra tions. This suggested that the in vivo variability was largely due to the toxic alterations of tissues, caused by carcinogen released at high rates. Pellets composed of beeswax:cholesterol in ratios of 1:1 to 1:9 showed markedly reduced rates of PCH release. At a ratio of 1:9, the overall release rate of benzo(a)pyrene was 1 /¿g/daycompared to 7 /¿g/dayfor pellets with a pure beeswax matrix [100 ¿igbenzo(a)pyrene pellets]. The variability of PCH release was simultaneously dimin ished, supporting the suspicion that it was a result of toxic tissue changes. Similarly reduced release rates could be obtained by adsorbing the PCH to charcoal particles. Studies with the promotor 12-O-tetradecanoylphorbol13-acetate showed a release rate of 1.6 /¿g/dayfrom pure beeswax (100 /¿g12-O-tetradecanoylphorbol-13-acetate per pellet). This rate may have to be diminished before promotion studies in the trachea! transplant model can be attempted. Our studies demonstrate that protracted PCH release from pellets can be achieved by using a beeswax: cholesterol matrix instead of a pure beeswax matrix or by adsorbing the PCH to charcoal particles. Such pellets release at fairly constant rates, with little pellet-to-pellet variability. Thus, the major problem of using the trachea! transplant model for quantitative tumor induction studies with PCH's appears to be resolved. 1 Research supported in part by the Environmental Protection Agency (1AG-D5-E681) and by the Department of Energy under contract with the Union Carbide Corporation. 2 Postdoctoral Investigator, Carcinogenesis Training Grant CA09285 from the National Cancer Institute. 3 Present address: Room A 322, Landow Building, National Cancer Insti tute, Bethesda. Md 20014. 4 To whom requests for reprints should be addressed, at Biology Division, Oak Ridge National Laboratory, P. 0. Box Y, Oak Ridge, Tenn. 37830. Received October 28. 1977; accepted February 13, 1978. INTRODUCTION We recently described the heterotopic trachéal transplant model as a new experimental system to study the develop ment of neoplastic disease in respiratory tract epithelium (6). We pointed out that this experimental model lends itself particularly well to investigation of the role of various host and environmental factors as modifiers of respiratory tumorigens since host and target organ can be independently manipulated and since initiators and carcinogens as well as promotors and cocarcinogens can be applied in known quantities to a narrowly defined target site. In this system the mucosa of s.c. trachéaltransplants is exposed directly to test substances contained in and released from an intraluminal, rod-shaped pellet. We have described the induction of neoplasms with several types of carcinogenic polycyclic hydrocarbons, the carcinogen dose-tumor re sponse relationships, and the time-dependent appearance of various presumed preneoplastic lesions following PCH5 exposure (3, 4,10). Since our first report of this experimen tal model (6), other laboratories have also used it with equal success (7,8). During our studies over the last 2 years, it became in creasingly clear that the ultimate utility of this experimen tal model depended largely on the development of a "drug delivery system" allowing protracted exposure at reproduc ible, fairly constant and controllable rates with a variety of chemicals. Clearly, it was necessary to investigate this component of the experimental model in greater detail than we had previously done. In the initial studies (6) the PCH's had been incorporated into pellets composed of a gelatin matrix that rapidly dissolved on introduction into the tra chéal lumen, releasing the total carcinogen dose within 5 to 7 min. Subsequently, pellets with a beeswax matrix were found to release the carcinogen more gradually (2, 4, 10). However, inconsistencies in PCH release rates in various studies prompted us to study the source of this variability and to look for means to correct this deficiency. The purpose of this report is to present our efforts at identifying the cause of the variability in the carcinogen delivery system used to induce trachéaltumors and to find means to reduce or eliminate it. In vitro and in vivo studies of the release characteristics of PCH's from beeswax pellets containing high and low concentrations of PCH's are de scribed. Them vitro studies were performed because of the speed of the assay and for investigation of PCH release independent of confounding biological variables. We report 5 The abbreviations used are: PCH, polycyclic hydrocarbon; TPA, 12-Otetradecanoylphorbol-13-acetate; BP, benzo(a)pyrene; DMBA, 7,12-dimethylbenz(a)anthracene; ¡.t.,intratracheal. 1376 CANCER RESEARCH VOL. 38 on April 13, 2017. © 1978 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Controlled Release of Carcinogens for Tumor Induction modifications of the pellet matrix, which, dependent on the pellet composition, effect markedly slower and more con sistent rates of PCH release than those obtained previously with a pure beeswax matrix. Finally, we describe release experiments with the classical promoting agent, TPA. These studies may be of interest not only to the student of respiratory tract carcinogenesis but also to investigators interested in protracted release of PCH's and TPA in other in vivo models of carcinogenesis. MATERIALS AND METHODS