We have used the technique of alkaline elution to determine the amount of nitrogen mustard (HN2)-induced DNA cross-linking in a murine fibrosarcoma tumor in vivo. Mice were either treated with HN2 directly or were pretreated with misonidazole (MISO) or diethyl maleate prior to injection with HN2. Two types of HN2-induced DNA lesions were detected, namely, proteinase K-sensitive and -resistant cross-links. Pretreatment with MISO did not appear to affect the ratio of the two types of lesion. In mice treated with HN2 alone, the amount of cross-linking reached a high level by 0.5 hr postinjection, after which these lesions were repaired, 62% of cross-links being removed between 0.5 hr and 6 hr postinjection. Pretreatment of mice with MISO resulted in substantial alterations in both the magnitude and time course of cross-linking during the first few hr after injection of HN2. Both MISO and diethyl maleate enhanced the number of cross-links formed at 0.5 hr postinjection. Furthermore, in MISO-pretreated mice, only 18% of the cross-links present at 0.5 hr had been removed by 6 hr postinjection. This early enhancement is possibly related to glutathione depletion resulting in reduced intracellular inactivation of HN2. Since repair processes were determined not to be saturated at the level of lesions under study, these data suggest that, in addition to the initial glutathione depletion resulting in an increased burden of damage, MISO may also inhibit DNA repair processes, possibly via a hypoxia-dependent interaction between MISO reduction products and DNA or repair enzymes. Assuming that DNA cross-linking is related to the cytotoxicity of HN2, these effects may account for the MISO enhancement of HN2 toxicity toward various biological systems which have been reported previously. It appears that chemosensitization may result from a variety of factors, with the relative importance of each factor depending on the particular drug being used.
This investigation was supported by USPHS Grants CA 26312 and CA 23270 awarded by the National Cancer Institute.