Abstract
Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS.
Highlights
Isopropyl methane sulfonate (IPMS), an alkyl sulfonate, is a potential genotoxic impurity (GTI) that can form as a byproduct during the synthesis of sulfonate salts when sulfonic acids react with isopropanol, a low molecular weight alcohol [1]
The mutant cell lines cover a variety of DNA repair/tolerance pathways (e.g., Base Excision Repair (BER), base excision repair; HEL, helicase; Nucleotide Excision Repair (NER), nucleotide excision repair; Non-Homologous End-Joining (NHEJ), non-homologous end-joining; Trans-Lesion Synthesis (TLS), translesion DNA synthesis; Homologous Recombination (HR), homologous recombination; DDC, DNA damage checkpoint)
The relative lethal concentration 50 (LC50) of methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS) in FANC-deficient cells was approximately 60%, while the value dropped to a www.impactjournals.com/oncotarget range of 10-40% in FANC-deficient cells treated with Isopropyl methanesulfonate (IPMS)
Summary
Isopropyl methane sulfonate (IPMS), an alkyl sulfonate, is a potential genotoxic impurity (GTI) that can form as a byproduct during the synthesis of sulfonate salts when sulfonic acids react with isopropanol, a low molecular weight alcohol [1]. IPMS-mediated DNA adduct www.impactjournals.com/oncotarget formation has been previously studied, it is important to determine its net biological effect (e.g. cytotoxicity and genotoxicity outcome), which is determined by the balance between the generation of DNA damage and the DNA repair efficiency. Understanding both the damage and repair aspects helps to more accurately interpret how individual alkylating agents induce genotoxicity. We conducted the DNA damage response (DDR) assay using isogenic chicken DT40 cell lines [12,13,14] to understand the repair or tolerant pathway activated in response to IPMS. The DDR assay, which examines cytotoxicity in DNA repair-deficient DT40 mutants versus the parental DT40 cells, is a rapid and simple method to evaluate the genotoxicity of xenobiotics
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