Depressed Incorporation of Thymidine-h3 into Deoxyribonucleic Acid following Administration of 7,12-dimethylbenz(alpha)anthracene.

In the experiments to be described, it was observed that the administration of 7,12-dimethylbenz(a)anthracene (7,12-DMBA) prior to the injection of tritiumlabeled thymidine (TdRH3) considerably depresses the incorporation of tritium into rat testis, intestine, and adrenal. Recovery from the effect of 7,12-DMBA occurs in intestine within 48 hr but in testis the inhibitory action of a single dose of 7,12-DMBA persists longer. Fractionation of the testis demonstrated that the decrease in tritium content is associated entirely with the perchloric acid-insoluble fraction. 7,12-DMBA is of special interest for several reasons. (1) A single dose evokes mammary cancer selectively, invariably, and rapidly in the rat under conditions which are easily satisfied.' (2) A single dose of the hydrocarbon invariably induces massive and selective necrosis of two of the zones of adrenal cortex2 of adult, but not of infant, rats.3 (3) In male rats, a solitary injection of 7,12-DMBA inflicts selective damage in the testicular cells which actively synthesize deoxyribonucleic acid (DNA) and only in cells of this class.4 Death of male germinal cells is not sudden, but occurs only after the injured cells have undergone one or several mitotic divisions. These spectacular biological effects of 7,12-DMBA made it desirable to investigate the influence of this compound upon the incorporation of thymidine into the DNA of testis and other tissues. Experimental.-Male rats of Sprague-Dawley strain were used exclusively; there were 4 animals in each group. Each experiment was repeated on 3 separate occasions with similar results. Tritium-labeled thymidine,5 specific activity of 3 c/mmol, was diluted with aqueous sodium chloride (0.9%), and a 1 ml dose, corresponding to approximately 1 gc/gm of body weight, was injected into a caudal vein. 7,12-DMBA, mp 122-3°, purified by Florisil chromatography and recrystallized from acetone-ethanol, was made up in lipide emulsion5 and injected once only into a caudal vein at various time intervals before or after the TdRH3. Infant rats, age 25 days weighing 52-70 gi, received 2 mg of 7,12-DMBA in 0.4 ml of emulsion; -adult rats, age 50 days weighing 200-220 gm, received 5 mg of 7,12DMBA in 1 ml of emulsion. The animals were killed by decapitation, and testis, adrenal, and a segment of terminal ileum were excised and weighed. For determination of the total volatile and nonvolatile radioactivity, each tissue was dried from the frozen state; the evaporated water was collected in a cold trap and counted directly, whereas the dry residue (triplicate samples for testis) was analyzed for tritium by the combustion procedure described previously.6 For determination of tritium in the acid-soluble and acid-insoluble fractions, the weighed testis was homogenized for 3 min in 4 ml of ice-cold 0.5 N perchloric acid containing 0.5 mg of nonradioactive thymidine per ml. All procedures after weighing were were carried out at a temperature of 40 or