GENETICS OF NEOPLASIA 255 



cross was intermediate between the F 1 and strain A, and the C57L backcrosswas inter- 

 mediate between the F 1 and strain C57L. From the divergence between the parental 

 strains and the excess of variance of the F 2 over that of the F l5 it was estimated that 

 these strains differed by a minimum of 4 pairs of genes for pulmonary tumors. 



This accurate quantitative measure of response of induced pulmonary tumors has 

 offered great advantages for genetic and carcinogenic studies. Because of this, presenta- 

 tion of some technical details is justified. A number of chemical carcinogens can be 

 used. Urethan is highly suitable if one wants a water-soluble carcinogen. It should 

 be injected intraperitoneally, however, because when injected intravenously the 

 anesthetic action is quick and there is danger of killing some of the animals. Crystalline 

 dibenzfa,/?] anthracene and methylcholanthrene are also highly satisfactory, and with 

 them a water dispersion satisfactory for injection can be prepared easily with a colloidal 

 mill.f Pulmonary-tumor response varies with size of the particles of the carcinogen in 

 the dispersion, and particle size varies from batch to batch, so not more than one batch 

 of dispersion should be used for an experiment. The intravenous injection of these 

 dispersions is most desirable since the crystals are filtered out in the capillaries of the 

 lung, and the pulmonary tumors are the only tumors induced. Mice can easily be 

 injected in the lateral tail vein with a 26 or 27 gauge, ^-inch, short-bevel needle. The 

 vein can be dilated with warm water or xylene, but usually this is not necessary. 

 Adult mice can easily tolerate \ ml. unless the material is toxic. Air bubbles must be 

 avoided. 



Although nitrogen and sulfur mustard are carcinogenic in inducing pulmonary 

 tumors, they are not recommended in genetic studies because of their toxicity and the 

 danger in handling them. Radiation would not be satisfactory since it does not 

 induce enough nodules. 



| Preparation of water dispersions of crystalline chemical carcinogens for intravenous 

 injection: 



Suggested concentrations: 1 mg./ml. of dibenz[a,/?]anthracene, methylcholanthrene, 

 benzypyrene, and so forth, in water plus 0.01 per cent aerosol plus 0.2 per cent Methocel, 

 with NaOH to pH 8. 



Suggested equipment: Colloidal mill (Eppenbach Processing Equipment, Manufactured 

 by Admiral Tool and Die, Inc., Long Island City, New York, U.S.A., Serial No. QV6- 

 53100). 



Preparation of basic solution : Put 2 g. of Methocel 4000 and 0. 1 g. aerosol in a large 

 beaker with 1000 ml. water. Heat, stirring constantly to dissolve Methocel. Remove from 

 heat and keep in refrigerator overnight. 



Preparation of dispersion: Put about 500 ml. basic solution in mill and start the mill. 

 Then add 1 g. carcinogenic crystals to the solution. Slowly turn the mill down to 0.005 

 inch and run at this setting for 5 minutes. Then turn mill to 0.003 inch and run for 5 

 minutes more. Remove carcinogenic dispersion and allow to stand for 5 minutes after 

 which pour off supernatant into a flask (about one half of dispersion). Put remainder of 

 dispersion back in mill using some of original basic solution to rinse flask. Turn mill to 

 0.002 inch and run for 2 minutes. Remove dispersion to a flask rinsing the mill with the 

 remainder of basic solution. Add supernatant and bring to pH 8 with 2n NaOH (about 

 2 drops). 



Throughout procedure stop mill often if necessary to prevent heating. 



