Abnormal base pairing under the influence of nitrogen mustard

Abstract

Cyclophosphamide, frequently used in tumor therapy, has proved to be mutagenic [l-4], but so far no clear evidence has been obtained in the host-mediated assay as to whether the substance causes point mutations [5]. Therefore we decided to approach the problem biochemically. Ludlum [6,7] has recently shown that the methylation of cytidine-diphosphate, or poly(C), and to a lesser degree that of the corresponding deoxyderivatives, alters the pairing characteristics of this base in such a way that in the transcription and/or polymerization process not only guanine, but also though to a smaller extent uracil and adenine are incorporated into the resulting polymer. Similar observations regarding the misreading of chemically altered poly(C) have been made by Singer and FraenkelConrat [8,9] and also by Means and Fraenkel-Conrat [IO]. On the basis of the results they obtained with several mutagens, these authors suggest that the substitution of position 3 of cytosine by an alkyl group might well represent a mutagenic event, inasmuch as methylated poly(C) shows appreciable ambiguity, thus leading to abnormal base pairing (with simultaneously diminished capacity for normal pairing with guanine). Since cyclophosphamide as such is not an alkylating agent, we utilized 0, /I’-dichlordiethylamine (nornitrogen mustard) (I) in our experiments. This compound has been postulated to be one of the active substances formed in the body after application of cyclophosphamide [ 111. Therefore, because of the alkylating and cross-linking properties of I [ 121, a strong diminution in the template function of poly(C) after incubation with I is by no means surprising. What could not be foreseen, however, was whether the treatment of poly(C) with such a relatively large molecule (large in comparison with methyl groups) would lead to abnormal base pairing. In fact, our findings have shown a very slight tendency towards the incorporation of LIMP, significantly higher than the “spontaneous” rate of abnormal base pairing with UMP, and increasing linearly with the time of incubation.