A method for hyperthermic treatment of mouse skin.

The Sencar mouse skin system is a recognized model for tumour initiation, promotion and progression. The current interest in the effect of hyperthermia on this multi-stage tumorigenesis model prompted the need for a technique to accurately heat a section of dorsal skin of a large number of mice for 30 min per heat treatment. In the technique described, experimental groups of 25 female Sencar mice were treated at 7-8 weeks of age under general methoxyflurane anaesthesia. Treatment consisted of the application of initiating and/or promoting agents with or without hyperthermia. For hyperthermic skin treatments, each group of mice was placed onto a platform in a water bath so that the dorsal skin of the mice was in contact with 44 degrees C temperature controlled water.

Hyperthermia and phorbol ester tumor promotion in mouse skin.

In a two-stage skin tumorigenesis protocol [7,12-dimethylbenz[a]anthracene (DMBA) initiation followed by twice weekly 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion], SENCAR mice developed an average of approximately 8.5 papillomas per animal. Hyperthermia treatments of the initiated skin (44 degrees C, 30 min) immediately before or after each TPA application (for 90 days) reduced papilloma frequency 80-90%. Animals whose initiated skin was made thermo-tolerant at the time of TPA application (by hyperthermia treatment 24 h prior to each application of promoter) showed slightly less protection (approximately 70% reduction in frequency). Multiple 44 degrees C hyperthermia treatments alone (27 X, twice a week) had no promoting activity in DMBA-initiated skin. The usual responses of skin to TPA promotion, including an increase in dark cells, epidermal thickening, reddening and erosion were all suppressed in animals treated with hyperthermia near the time of TPA application. The effect of hyperthermia on tumorigenesis was at the promotion stage and the survival of initiated cells was not affected, since the normal number of papillomas was produced when TPA promotion was delayed until after the multiple (27 X, twice a week) hyperthermia treatments were completed. Hyperthermia treatments (44 degrees C, 30 min, twice weekly for 90 days) given near the time of TPA application also suppressed the incidence of carcinomas appearing within 300 days. About 40% of the DMBA-initiated, TPA-promoted animals developed a carcinoma, compared with only approximately 10% of a similar group which received hyperthermia treatments near each TPA application. Papillomas appearing in spite of hyperthermia treatments during promotion were not more likely to progress into carcinomas than those appearing in unheated animals. Such hyperthermia treatments given to animals bearing pre-existing papillomas did not markedly alter the subsequent development of carcinomas compared with unheated controls. The results demonstrated that 44 degrees C hyperthermia applied near the time of TPA promotion acted as a powerful antipromoter and suppressed the appearance of both papillomas and carcinomas, apparently by acting at an early stage of promotion.

Fluorometric method for rapid detection of DNA strand breaks in human white blood cells produced by low doses of radiation.

DNA strand breaks can be detected with great sensitivity by exposing crude cell lysates to alkaline solutions and monitoring the rate of strand unwinding. As little as one strand break per chromosome can be detected. Previous methods for measuring strand unwinding have required physical separation of single- from double-stranded molecules. We now describe conditions under which unwinding can be monitored directly using a fluorescent dye, thus greatly simplifying the analysis. Breaks due to irradiation of blood samples by 60Co gamma-rays at doses as low as 0.05 to 0.1 gray (5 to 10 rads) were detectable. Rapid rejoining of strand breaks during in vitro incubation at 37 degrees could readily be observed following a dose of one gray. Since the procedure is very rapid and cells can be analyzed directly without the requirement for culturing or radiolabeling, the procedure could be useful in cancer chemotherapy if in vivo damage is to be monitored or for testing the in vitro sensitivity of cells to drugs.