Craig J. Boreiko

Initiation of solC3H10T12 cell transformation by formaldehyde

Abstract The effects of formaldehyde were evaluated in the C3H 10T 1 2 Cl 8 cell transformation system. Treatment of the cells with 0.1–2.5 μg/ml of formaldehyde alone did not result in significant rates of transformation. If formaldehyde treatment was followed by continuous treatment with 0.1 μg/ml of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), transformed foci were produced. Methanol and formic acid lacked significant transforming activity under either treatment regimen. The results suggest that formaldehyde is an initiating agent for C3H 10T 1 2 Cl 8 transformation. The fact that some compounds may act solely as initiators should be considered when this transformation system is used to study chemicals which may interact with cells by mechanisms similar to that of formaldehyde.

Mutagenic and Carcinogenic Effects of Formaldehyde

Formaldehyde is an important commodity chemical with widespread and diverse industrial applications (1,2). Formaldehyde production in the United State has been steadily increasing for a number of years, and was close to 3 million tons in 1978 (1). World-wide formaldehyde production approached 12 million tons (1). Approximately 50% of the formaldehyde produced in the United States is utilized in urea-formaldehyde and phenol-formaldehyde resin production (1,2). These resins are used in turn for the manufacture of particle board, plywood and home-insulation foams. Formaldehyde resins are also used as molding compounds for the manufacture of dinnerware, appliances and telephones. Other uses of formaldehyde include the production of herbicides, pharmaceuticals and permanent-press clothing. A significant portion of the U.S. work force is thus involved in the manufacture and utilization of formaldehyde or formaldehyde based products (3).

Development of a tracheal implant xenograft model to expose human bronchial epithelial cells to toxic gases.

A tracheal implant model was developed which enabled exposure of differentiated normal human bronchial epithelial cells (NHBE) to a single exposure of a model toxic gas (formaldehyde). NHBE cells were grown in vitro in explant culture under defined serum-free conditions from previously frozen bronchial segments, harvested, and used to repopulate de-epithelialized rabbit tracheas. Rabbit tracheal segments repopulated with NHBE cells were implanted into the subcutis of congenitally athymic nude mice. Following graft vascularization, the xenografted NHBE cells differentiated and formed a mucociliary epithelial surface which lined approximately 50% of the surface of the implant lumen. Eight weeks post-implantation both ends of the implanted grafts were cannulated, and formaldehyde (HCHO) vapor (0, 6, or 15 ppm) in humidified air was passed through the tracheal lumens. Representative epithelial surfaces were examined by light and scanning electronmicroscopy, and autoradiography prior to, immediately after, and 48 hr following a 1-hr exposure to the test vapors. Light microscopic examination of implant sections immediately following exposure to 6 and 15 ppm HCHO detected cessation of ciliary activity, which recovered by 48 hr postexposure. Scanning electron microscopic examination of the epithelial surface demonstrated mild morphologic changes, restricted to those implants exposed to 15 ppm. Findings immediately following HCHO exposure included swelling and exfoliation of individual cells, deciliation, and mucus release. Changes present after 48 hr included presence of flattened cells with few short microvilli and focal increase in the number of S-phase nuclei in the basal epithelium. These results demonstrate the utility of tracheal implants for single short-term exposure of differentiated human bronchial epithelial cells to gaseous agents. This system should prove useful for mechanistic studies of human upper airway toxicity.

The use of tracheal implants in toxicology and carcinogenesis research

Tracheal implants have served as an important experimental pathology tool with which to study the toxic and/or carcinogenic effects of chemicals upon upper respiratory tract epithelium. Initial studies with this method utilized heterotopic rat tracheal transplants which were exposed to compounds of interest, and assessed for toxic and/or carcinogenic endpoints. Grafts containing rodent tissue have proved useful for studying the cellular and biochemical features of neoplastic progression at different time intervals following in vivo exposure to carcinogens. More recent studies have utilized epithelial denuded tracheal implants inoculated with respiratory cell populations, and xenografted into immunodeficient nu/nu mice. This technique permits the study of airway epithelium from a variety of species, including man. The advent of molecular pathology techniques such as in situ hybridization will further expand the uses of tracheal implant technology for studies with xenografted human tissues. Such implants should prove useful for the examination of species- and tissue-specific characteristics of growth and differentiation by providing a bridge between cell culture and whole animal studies.

Lack of di-(2-ethylhexyl) phthalate activity in the C3H10T12 cell transformation system

The widely used plasticizer and rodent carcinogen di-(2-ethylhexyl) phthalate (DEHP) was examined for activity in the C3H 10T 1 2 murine fibroblast cell transformation system. Treatment with DEHP or its metabolite, mono-(2-ethylhexyl) phthalate, did not produce oncogenic transformation, initiate the process of transformation in cultures treated with a tumour promoter or promote the process of transformation in cultures pretreated with a chemical carcinogen. These findings are consistent with the suggestion that the carcinogenicity of DEHP is mediated by an indirect mechanism and not by covalent interaction of DEHP with DNA.

Differential effects of tumour promoters on the growth of normal human bronchial epithelial cells and human lung tumour cell lines

The effects of the tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on the colony-forming efficiency and growth of normal human bronchial epithelial (NHBE) cells and five lung squamous carcinoma cell lines were compared in medium containing 1% foetal bovine serum. TPA (0.1-5.0 ng/ml) inhibited the growth of NHBE cells and one carcinoma cell line, while four of the five carcinoma lines were less sensitive to the growth inhibitory properties of TPA but were slightly inhibited at higher TPA concentrations. The responses of NHBE cells and carcinoma cells to TPA, and the related compounds, mezerein, 4-O-methyl TPA, and phorbol were then compared in serum-free medium. In general, the removal of serum from the medium increased the differences in the responses to TPA between normal and tumour cells. Two carcinoma lines inhibited by TPA in 1% serum were stimulated by TPA in the absence of serum. Mezerein and, to a lesser extent, 4-O-methyl TPA also produced differential responses in colony-forming efficiencies between tumour lines and NHBE cells. Phorbol had no effect on either NHBE cells or on carcinoma cell lines. The relative insensitivity of carcinoma cell lines to the growth inhibitory effects of tumour promoters is consistent with the hypothesis that tumour promotion involves selection against normal cells to permit clonal expansion of preneoplastic or neoplastic cell types.

Methodology for cell transformation assays with C3H/10T1/2 mouse embryo fibroblasts

Basic cell culture conditions required for execution of cell transformation studies with C3H/10T1/2 mouse embryo fibroblasts are described. These protocols permit assessment of the ability of chemicals to convert nontumorigenic C3H/10T1/2 cells to a tumorigenic phenotype characterized by altered controls upon in vitro cell division. Recent assay modifications that enhance the sensitivity of this focus formation assay and permit the study of multistage transformation processes are noted.