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 N 5-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.

Liquid Chromatography–Mass Spectrometry Screening of Cyclophosphamide DNA Damage In Vitro and in Patients Undergoing Chemotherapy Treatment

“…The MS 2 spectra collected were consistent with the ones previously reported by Chen and colleagues. 10 Furthermore, the MS 3 spectra collected in our work provided further confirmation of the chemical nature of the modifications. Indeed, the appearance of one of the nucleosides or one of their fragments (Table 1) gives insights into where the modification is located.…”

“…Specific DNA adducts generated by CPA in vitro and in vivo have been previously evaluated with liquid chromatography–mass spectrometry (LC–MS) methods (Figure ). − …”

In Vitro Evaluation of DNA Damage Effect Markers toward Five Nitrogen Mustards Based on Liquid Chromatography–Tandem Mass Spectrometry

“…Kumar et al once showed that as per 12 and 24 h after exposure to HN2 in hairless mice, obvious DNA oxidative damage, lipid peroxidation, and inflammation were observed. However, for HN2-treated female mice, the mono-alkylated N 7 -guanine adduct reached a maximum level within 6 h in the liver, lung, and spleen . It is thus speculated that, for these three NMs, HN1 with moderate cytotoxicity was more likely to promote endogenous acetaldehyde production and inflammation than less toxic HN3.…”

“…However, for HN2-treated female mice, the monoalkylated N 7 -guanine adduct reached a maximum level within 6 h in the liver, lung, and spleen. 32 It is thus speculated that, for these three NMs, HN1 with moderate cytotoxicity was more likely to promote endogenous acetaldehyde production and inflammation than less toxic HN3. Nonetheless, HN2 with the highest toxicity may have a stronger direct damage effect on cells, but a weaker lipid peroxidation and inflammatory response.…”

“…Even though the DNA lesions caused by exogenous exposure of NMs have been directly assessed via the exogenous DNA adducts, such as the NM conjugated DNA interstrand cross-linking, , DNA–protein cross-linking, and the formation of the apurinic site, the effect markers may be a better choice to reflect its overall toxic effect consisting of multiple DNA damage pathways. To our knowledge, it is the first simultaneous measurement of a DNA damage effect panel consisting of five DNA damage effect markers and three DNA epigenetic modifications to evaluate the general alterations of DNA endogenous damage caused by NMs, and to unveil the relationship between the amount of DNA adducts and the level of cytotoxicity in vitro.…”