Synergistic Action of Nitrogen Mustard and Radiation in Microorganisms.

It is likely that the production of structural defects in DNA is a prominent molecular event relevant to cellular inactivation by such diverse agents as X rays, ultraviolet light (UV), and the radiomimetic alkylating agent, nitrogen mustard. -3 Although the chemical nature of these defects and the reaction mechanisms involved in their formation are undoubtedly different for each agent, the existence of an important cellular target makes a comparison of their respective inactivation kinetics especially interesting,4 and also raises the possibility that synergistic interactions5 might occur among them.6 Recent studies have indicated that certain chemical modifications of the structure of DNA, such as the incorporation of halogenated base analogues, result in an increased cellular response to both X rays and ultraviolet light.7-'0 Furthermore, bacteria containing sublethal UV photoproducts, a large fraction of which are photoreactivable and presumably in DNA, have an increased sensitivity to X rays."', 12 Since it also has been shown that nitrogen mustard (HN2) can produce interstrand cross links and chain breaks in DNA in vitro,3' 14 one might reasonably expect that any such structural defects produced sublethally in vivo could also serve to enhance the killing effect of ultraviolet and ionizing radiation. First, this paper describes our technique for obtaining reproducible HN2 doseeffect curves for bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) as a function of the initial HN2 concentration in the cell suspensions. For haploid and diploid yeast the resulting HN2 curves are similar in shape to the corresponding X ray survival curves, that is, exponential for haploid and sigmoid for diploid.6 Second, interaction experiments have shown that HN2 and 2537 A ultraviolet light act synergistically in the inactivation of haploid and diploid yeast, and E. coli B/r. On the other hand, while HN2 and X rays interact synergistically in B/r and diploid yeast, no such interaction occurs in haploid yeast. Materials and Methods.-Cultures of haploid and diploid yeast (Saccharomyces cerevisiae, SC-7 and SC-6) were grown for 6-8 days on potato-dextrose-agar (Difco) slants; E. coli B and B/r were grown for 18-20 hr either in nutrient broth or 1% peptone. The cells were then harvested, washed, and suspended in M/15 phosphate buffer (pH = 7.0) prior to treatment with 150 kv X rays (HVL = 1 mm Al), 2537 A ultraviolet light, or methyl bis (,B chloroethyl) amine hydrochloride (HN2). The suspensions were aerated by bubbling with sterile, water-saturated air during all X irradiations in order to maintain a maximum radiobiological oxygen effect. Yeast survivors were scored by their ability to form visible colonies on potato-dextrose-agar plates following 4 days' incubation at 28°C; the coli were plated on nutrient (salt) agar, and visible colonies counted after 18 hr incubation at 370C. Irradiation and calibration techniques for the X ray and UV sources have already been described;15 the X ray dose rate was 4.37 kilorads/min, and the incident UV flux (90% at 2537 A) was 20 ergs/mm2/sec. Inactivation by HN2 was effected in the following manner: a concentrated aqueous solution of nitrogen mustard was made by dissolving a known amount of the desiccated hydrochloride salt in a known volume of the phosphate buffer. One ml of this solution was then diluted with 9 ml of cell suspension within 20 sec after preparation, producing the initial "exposure" concentration. The suspensions were agitated continuously at room temperature (240C), and samples were