Analysis of Different Fates of DNA Adducts in Adipocytes Post-sulfur Mustard Exposure in Vitro and in Vivo Using a Simultaneous UPLC-MS/MS Quantification Method

Abstract

Sulfur mustard (SM) is a powerful alkylating vesicant that can rapidly penetrate skin, ocular, and lung bronchus mucous membranes and react with numerous nucleophiles in vivo. Although the lesion mechanisms of SM remain unclear, DNA damage is believed to be the most crucial factor in initiating SM-induced toxicity. Four major DNA adducts were identified for retrospective detection and DNA lesion evaluation, namely, N(7)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N(7)-HETEG), bis(2-ethyl-N(7)-guanine)thioether (Bis-G), N(3)-(2-hydroxyethylthioethyl)-2'-adenine (N(3)-HETEA), and O(6)-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (O(6)-HETEG). Because of previous observations that the levels of SM-DNA adducts were relatively higher in adipose-rich organs, such as the brain, we focused on the in vitro and in vivo fates of the DNA adducts in exposed adipocytes. A UPLC-MS/MS method developed in our laboratory was used to profile the N(7)-HETEG, Bis-G, and N(3)-HETEA levels in human mature adipocytes (HA-s) that had differentiated from human subcutaneous preadipocytes (HPA-s). This method was also used to profile three other cell lines related to the targeting of major tissues, including human keratinocytes (HaCaT), human hepatocytes (L-02), and human lung fibroblasts (HLF). Long-lasting adduct persistence and a high proportion of Bis-G were found in exposed adipocytes in vitro. The survival properties of exposed adipocytes were also tested. At the same time, the fate of SM-DNA adducts in vivo was characterized using a rat model exposed to 1 and 10 mg/kg doses of SM. The level of DNA adducts in the exposed adipose tissue (AT) was much lower than those in other organs studied in our previous work. The adduct persistence behavior was observed in AT with an extremely high proportion of Bis-G, which was higher than N(7)-HETEG. In light of these results, we suggest that an adipose-rich environment may promote the formation of Bis-G and that adipocyte-specific DNA repair mechanisms may result in adduct persistence and the survival of adipocytes after SM exposure. These conclusions should be further investigated.

Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of Bis(2-chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

“…56 Wang et al reported that the SM (100 μ M) also forms the structurally related N7-[2-[(2-hydroxyethyl)thio]ethyl]-guanine (N7-HETEG) and bis[2-(guanin-7-yl)ethyl]-sulfide (Bis-G), among other adducts, as the biomarkers for DNA damage in rodents and human keratinocytes, hepatocytes and lung fibroblasts. 64 Very high levels N7-HETEG and G-SM-G cross-link (> 4 adducts per 10 5 bases) were formed in all cell lines. The t 1/2 values of N7-HETEG and its cross-link showed no significant differences among the liver, lungs, and skin-derived cells.…”

“…The chemistry and reactivity of nitrogen 8-10, 18, 20, 24, 47-56 and sulfur 44, 57-64 mustards with nucleosides and DNA in vitro is well studied. There are also numerous studies that examine the mustard derived DNA adducts from treated cell culture and animals, 46,48,50,54,56,59,64,65 including white blood cells of patients on chemotherapeutic regimens that include a nitrogen mustard.…”

“…53, 57 A variety of methods have been used to identify these adducts including 32 P-postlabeling, 47, 58 co-chromatography by HPLC with authentic standards, 8, 9, 24, 48, 57 and mass spectrometry. 18, 20, 49-52, 56, 59, 64 Recently, tandem mass spectrometry with stable isotope dilution has been employed to identify and quantitate some of these DNA adducts. 53, 54, 61-63 Collectively, the studies indicate that these electrophiles react predominantly at the N7 atom of dG, although N3-dA modification can also be significant.…”

A mass spectrometric platform for the quantitation of sulfur mustard-induced nucleic acid adducts as mechanistically relevant biomarkers of exposure

“…Follow-up studies focused on urinary excretion of metabolites and adduct distributions in different rodent organs (Yue et al 2015;Zhang et al 2014). In addition, these authors analyzed adduct levels in adipocyte cell lines and adipose tissue, suggesting a depot effect of SM in lipidrich environments (Wang et al 2015).…”

“…Despite this, SM was broadly used in the Iran-Iraq War (1980)(1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988) and even recently in asymmetric conflicts, such as in Syria and Iraq (Dons 2013;OPCW 2017;Thiermann et al 2013). Due to ease of chemical synthesis as well as potentially undestroyed residual stockpiles in conflict areas, it may also be accessible for terroristic groups (Balali-Mood and Hefazi 2005;Wang 2010). Therefore, SM still represents a realistic threat, both for civilians and soldiers, which calls for reliable, specific, and sensitive exposure assessment.…”