Studies of prostaglandins in rat mammary tumors induced by 7,12-dimethylbenz(a)anthracene.

a modification of the procedure described by Huggins et aL (3). A lipid emulsion (0.2 ml) of 1% (w/v) DMBA was injected into the caudal vein of animals at ages of 50, 53, and 56 days. Fifty mg of DMBA were dissolved in 1.5 ml of corn oil, and the mixture was warmed and then stirred with a Vortex mixer until a clear solution was obtained. The final emulsion was obtained by mixing 3.5 ml of fresh rat serum with this solution on a Vortex mixer. Prostaglandin reference standards, PGE1 and PGE2 (95% pure) and arachidonic acid were obtained from the Lipids Preparation Laboratory of The Hormel Institute. Radioactive arachidonic acid (5,8,1 1,14-eicosatetraenoic 4C) specific activity, 55 mCi/mmole, was obtained from Dhom Products, Ltd., North Hollywood, Calif. Prostaglandin Biosynthesis. Biosynthesis was assayed by a modification of the procedure described by Lapidus et a!. (10). The tissue (1 g) was homogenized in 10 ml of 0.2 M Tris-HC1 buffer, of 1 mM glutathione, adjusted to a fmal pH of 8 with HC1.The homogenates wereincubatedwith the radioactive substrates in an atmosphere of air for 1 hr at 37° with shaking. The reaction was stopped by adjusting the pH of the mixture to 3 with HC1. The tissue was removed by either filtration or centrifugation, and the tissue-free solution was extracted with 3 volumes of ethyl acetate . The combined ethyl acetate extracts were reduced in volume by evaporation to approximately 20 ml and extracted twice with 10 ml of 0.2 M phosphate buffer at pH 8. This extract was adjusted to a pH of 3 with HC1 and extracted twice with equal volumes of dichioromethane. The dichloromethane extracts were evapo rated under a negative pressure, and the residue was dissolved in a known volume of 95% ethanol. Aliquots of the ethanol solution were used for identification and isolation of the prostaglandin by TLC. The percentage conversion of the fatty acid substrate was determined by measurement of the radioactivity of the isolated prostaglandins in a toluene scintillation solution of PPO and POPOP, with a Packard Tri-Carb Model 3320 scintillation spectrometer. TIC. The isolation and identification of individual prostaglandins were carried out by TLC by a combination of siicic acid and argentation techniques, as described by Green and Samuelsson (2) and Ramwell and Daniels (14). Radioimmunoassay of Tissue Prostaglandins. The mam mary tissues were homogenized 3 times in 10-mi volumes of ethanol. The combined ethanol extract was evaporated to dryness and the residue was redissolved in an equal volume of ethyl acetate. The prostaglandins were recovered with the use of phosphate buffer, dichloromethane, and ethanol as de scribed in the biosynthesis section (9, 13). The radioimmuno