Cytotoxic effect of 1,3 bis (2-chloroethyl)-N-nitrosourea at elevated temperatures: Arrhenius plot analysis and tumour response

Abstract

The effect of hyperthermia on the cytotoxicity of 1,3-bis-(2-chloroethyl)-N-nitrosourea (BCNU) was investigated in vitro and in vivo. Tumour cells were early-generation isotransplants of a spontaneous C3Hf/Sed mouse fibrosarcoma, FSa-II. For in vitro studies, single cell suspensions containing 1.0 x 10(6) cells/ml were treated in a water bath where a desired temperature was maintained by a constant-temperature circulator. Cell survival was determined by lung colony assay immediately thereafter. For in vivo studies the tumour cell suspensions were transplanted into the dorsal site of the C3Hf/Sed mouse foot. Tumours were treated by immersing animal feet into a constant-temperature water bath when tumours reached an average diameter of 4 mm (35 mm3). The tumour growth (TG) time or the time for one-half of the treated tumours to reach 1000 mm3 from initial treatment day was used as an endpoint. BCNU dose-cell survival curve at 37 degrees C was exponential with a D0 of 1.1 microgram/ml. Dose-cell survival curves at 37-43 degrees C were determined as a function of treatment time at pH 6.7 and 7.4. BCNU of 1 microgram/ml was added immediately before treatment. The slope of the survival curve became steeper with increasing temperature, indicating that the cytotoxic effect of BCNU was enhanced by hyperthermia. The Arrhenius plot analysis showed that activation energies at pH 6.7 and 7.4 were 53 and 51 kcal/M, respectively (no significant difference). Of interest in this analysis was that the Arrhenius plot did not show a breaking point which has been observed for other agents. Further investigation demonstrated that the decomposition of BCNU, which has been reported to be essential for production of reactive intermediates, occurred in aqueous medium at elevated temperatures. The magnitude of this decomposition depended on treatment temperature. As a result, preheated BCNU became less cytotoxic with an increase in preheating temperatures. The activation energy for this decomposition was about one-half of the activation energy for BCNU cytotoxicity. Studies in vivo indicated that the effect of BCNU was enhanced with increasing temperatures, and the enhancement was greatest when BCNU was administered i.p. immediately before hyperthermia. A glucose dose of 5 g/kg administered i.p. 60 min before hyperthermia further enhanced the antitumour effect of BCNU.