Kinetics of DNA Adducts and Abasic Site Formation in Tissues of Mice Treated with a Nitrogen Mustard

Abstract

Nitrogen mustards (NM) are an important class of chemotherapeutic drugs used in the treatment of malignant tumors. The accepted mechanism of action of NM is through the alkylation of DNA bases. NM-adducts block DNA replication in cancer cells by forming cytotoxic DNA interstrand cross-links. We previously characterized several adducts formed by reaction of bis(2-chloroethyl)ethylamine (NM) with calf thymus (CT) DNA and the MDA-MB-231 mammary tumor cell line. The monoalkylated N7-guanine (NM-G) adduct and its cross-link (G-NM-G) were major lesions. The cationic NM-G undergoes a secondary reaction through depurination to form an apurinic (AP) site or reacts with hydroxide to yield the stable ring-opened N⁵-substituted formamidopyrimidine (NM-Fapy-G) adduct. Both of these lesions are mutagenic and may contribute to secondary tumor development, a major clinical limitation of NM chemotherapy. We established a kinetic model with NM-treated female mice and measured the rates of formation and removal of NM-DNA adducts and AP sites. We employed liquid chromatography-mass spectrometry (LC-MS) to measure NM-G, G-NM-G, and NM-Fapy-G adducts in liver, lung, and spleen over 168 h. NM-G reached a maximum level within 6 h in all organs and then rapidly declined. The G-NM-G cross-link and NM-FapyG were more persistent with half-lives over three-times longer than NM-G. We quantified AP site lesions in the liver and showed that NM treatment increased AP site levels by 3.7-fold over the basal levels at 6 h. The kinetics of AP site repair closely followed the rate of removal of NM-G; however, AP sites remained 1.3-fold above basal levels 168 h post-treatment with NM. Our data provide new insights into NM-induced DNA damage and biological processing in vivo. The quantitative measurement of the spectrum of NM adducts and AP sites can serve as biomarkers in the design and assessment of the efficacy of novel chemotherapeutic regimens.

Figure 1.

EIC of NM-DNA adduct formation in the liver of female C57BL/6NJ mice injected i.p. with saline or NM (3 mg/kg). Animals were sacrified 6 h post-treatment.

Figure 2.

Time course of the NM-DNA adduct levels in mouse DNA after treatment of NM. Female C57BL/6NJ mice were injected i.p. with NM (3 mg/kg) and sacrificed after 6, 24, 48, 72, and 168 h. Control mice were injected i.p. with saline and sacrificed at T0 h. Adduct levels in liver, spleen, and lung DNA are expressed as adducts per nts (mean ± SEM, n = 5). The dashed lines represent the background signals of the MS³ transitions for NM-G and NM-Fapy-G. The background signal for G-NM-G is below the background signal of NM-G and not presented.

Figure 3.

(A) EIC of AP site levels and internal standard in female mouse liver DNA after treatment of NM (3 mg/kg) at 6 h and 168 h. Control mice were injected i.p. with saline and sacrificed at T0 h. (B) One-phase decay model was applied to estimate the half-life of AP site removal (mean ± SD, n = 5 animals per data point). The decay plateau was constrained to the basal level of AP sites and shown as a dashed line. (C) Levels of AP sites in NM-treated mice at 6 and 168 h versus control mice (mean ± SD, n = 5 animals per data point). A Welch’s t-test for unequal variances was applied to compare the significant difference between the treated group at 6 and 168 h and the untreated control group. **** p < 0.0001 and ** p = 0.0039.

Scheme 1.

Mechanism of NM-DNA adduct and AP site formation.