ANALYSIS OF CYCLOPHOSPHAMIDE-INDUCED N5-NITROGEN MUSTARD FORMAMIDOPYRIMIDINE ADDUCT IN DRIED BLOOD SPOT USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Cyclophosphamide is one of the nitrogen mustard agents that is frequently used in cancer chemotherapy. Nevertheless, long-term use of high dosage cyclophosphamide has been proven to increase the risk of secondary cancer. This can be traced by the mutagenic DNA adduct formation, for instance, N5-nitrogen mustard formamidopyrimidine (NM-Fapy-G). Therefore, it may serve as one of the secondary cancer biomarkers in patients receiving cyclophosphamide. Several NM-Fapy-G analysis methods employ Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) developed by experts. However, it was discovered not applicative for patients because cells and tissues were utilized as the biospecimens. Therefore, this summary is presented to emphasize the idea of adopting Dried Blood Spot (DBS) as the blood bio sampling method, DNA extraction and hydrolysis method that is suitable for enriching NM-Fapy-G adduct; and method that is proper for NM-Fapy-G analysis. Based on the literature study, DBS has been proven beneficial for this analysis; DNA can be extracted from the DBS cards using the QIAamp DNA Mini Kit. Therefore, research with a little bit of adjustment can be applied for NM-Fapy-G analysis.

Determination of nitrogen mustard metabolites in urine using high-performance liquid chromatography and high-resolution mass spectrometry

Detection of biologically active components in mixtures of complex composition which include biological fluids (blood, urine, etc.) is the most difficult problem of the analytical chemistry and requires the use of modern highly informative research methods. One of the most effective and universal ways to study the structure of unknown substances is the method of liquid chromatography- high resolution mass spectrometry that combines the possibility of highly selective separation of the mixtures under study, the reliable detection of unknown substances and high sensitivity of the procedure. We propose a method for the simultaneous extraction of highly polar biomarkers of nitrogen mustard — N-triethanolamine (TEA), N-ethyl diethanolamine (EDEA) and N-methyldiethanolamine (MDEA) — from urine with subsequent determination by HPLC and detection by high resolution tandem mass spectrometry. The mass spectra of fragmentation of protonated molecular ions of TEA, EDEA, and MDEA have been studied and possible structural formulas of the fragment ions are given. The sample preparation of urine and mass spectrometric detection in the multiple reaction monitoring mode were optimized. The five-fold dilution with deionized water was chosen as a method of urine sample preparation for analysis. Separation of the components was performed in the reversed-phase chromatography mode with retention times for TEA, EDEA, and MDEA of 2.00, 2.05, and 1.92 min, respectively. The time required to complete all steps of the urine sample analysis did not exceed 25 min. The detection limits in urine were 1 ng/ml for TEA and 2 ng/ml for EDEA and MDEA. The developed approach provides determination of the fact of application of specific nitrogen mustard in enquiry of possible exposure of living organism to the blister agents.

Mast cells promote nitrogen mustard mediated toxicity in the lung associated with proinflammatory cytokine and bioactive lipid mediator production

Sulfur mustard (SM) has been widely used as a chemical warfare agent including most recently in Syria. Mice exposed to SM exhibit an increase in pro-inflammatory cytokines followed by immune cell infiltration in the lung, however, the mechanisms leading to these inflammatory responses has not been completely elucidated. Mast cells are one of the first responding innate immune cells found at the mucosal surfaces of the lung and have been reported to be activated by SM in the skin. Therefore, we hypothesized that nitrogen mustard (NM: a surrogate for SM) exposure promotes activation of mast cells causing chronic respiratory inflammation. To assess the role of mast cells in NM mediated pulmonary toxicity, we compared the effects of NM exposure between C57BL/6 and B6.Cg-KitW-sh/HNihrJaeBsmJ (KitW-sh; mast cell deficient) mice. Lung injury was observed in C57BL/6J mice following NM exposure (0.125 mg/kg) at 72 hrs, which was significantly abrogated in KitW-sh mice. While both strains exhibited damage from NM, C57BL/6J mice had higher inflammatory cell infiltration and more elevated prostaglandin D2 (PGD2) present in bronchoalveolar lavage fluid compared to KitW-sh mice. Additionally, we utilized murine bone marrow derived mast cells to assess NM-induced early and late activation. While NM exposure did not result in mast cell degranulation, we observed an upregulation in PGD2 and IL-6 levels following exposure to NM. Results suggest that mast cells play a prominent role in lung injury induced by NM and may contribute to the acute and potentially long-term lung injury observed caused by SM.

Alkylating Agents, the Road Less Traversed, Changing Anticancer Therapy

: Cancer is considered one of the gruelling challenges and poses a grave health hazard across the globe. According to the International Agency for Research on Cancer (IARC), new cancer diagnoses increased to 18.1 million in 2018, with 9.6 million deaths, bringing the global cancer rate to 23.6 million by 2030. In 1942, the discovery of nitrogen mustard as an alkylating agent was a tremendous breakthrough in cancer chemotherapy. It acts by binding to the DNA, and creating cross linkages between the two strands, leading to arrest of DNA replication and eventual cell death. Nitrogen lone pairs of ‘nitrogen mustard’ produce an intermediate 'aziridinium ion' at molecular level, which is very reactive towards DNA of tumour cells, resulting in multiple side effects with therapeutic consequences. Owing to its high reactivity and peripheral cytotoxicity, several improvements have been made with structural modifications for the past 75 years to enhance its efficacy and improve the direct transport of drugs to the tumour cells. Alkylating agents were among the first non-hormonal substances proven to be active against malignant cells and also, the most valuable cytotoxic therapies available for the treatment of leukaemia and lymphoma patients. This review focus on the versatile use of alkylating agents and the structure activity relationship (SAR) of each class of these compounds. This could provide an understanding for design and synthesis of new alkylating agents having enhanced target specificity and adequate bioavailability.