The Influence of Iron-dextran Complex on the Strain L Fibroblast

Iron-dextran complex has been found to be carcinogenic in animals. Richmond (9, 10), Haddow (3), and Haddow and Horning (4) have reported that subcutaneous injections of massive doses of this complex in the rat and mouse have given rise to sarcomas originating from iron-laden connective and reticuloendothelial tissues. It was thought that it would be interesting to grow the mouse subcutaneous fibroblast in culture in the presence of massive doses of the iron-dex-tran complex. In such cultures the morphology, growth, and metabolism, particularly the respiration , could be studied. The experimental evidence to be reported here are the changes which have occurred in these cultures during the first 3 months in which they were grown in medium containing iron-dextran complex. METHODS Cultures: Stock cultures of the mouse subcutane-ous fibroblast, strain L, clone 929, were grown in Roux flasks under standard culture conditions (6). The nutrient medium used was a modified Eagle's medium (6) plus 2 per cent calf serum and 5 per cent Bacto-peptone. 0.1 milliliter of iron dextran complex, Imferon, (equivalent of 500 /~g Fe) per milliliter was added to the nutrient fluid. The fluid was changed twice weekly. The cell population was maintained at approximately 104 to 105 cells per milliliter. For detailed studies, the cells were grown in test tube cultures. Cells from Roux flasks were trypsinized (~50 Difco), washed, and resus-pended in nutrient fluid containing the same amount of iron-dextran as in the stock cultures. A tricarboxylic acid supplement (1) was added to the medium in order to insure constant maximal respiration during the culture period. Two milliliters of this cell suspension, containing approximately 100,000 cells per milliliter, were introduced into regular test tubes. duplicate test tube cultures were done daily. Each culture was drained and 2 milliliters of 0.25 per cent trypsin (~50 Difco) were added. After 5 minutes the test tube was shaken to disperse the cells. A sample was examined microscopically to be sure that the cells had been dispersed. The cell concentration was then determined using a Ljungborg celloscope. Preparations for Cytological Study: In order to obtain cells for cytological study, coverslips were introduced into both test tube cultures and Roux flasks at the time the cultures were set up (6). Cells adhered to the upper surface of the cover-slips. The coverslips were removed from the cultures at various intervals, washed in balanced salt solution, fixed in formalin for 5 minutes, and …