Repair of sulfur mustard-induced DNA damage in mammalian cells measured by a host cell reactivation assay

Abstract

DNA damage is thought to be the initial event that causes sulfur mustard (SM) toxicity, while the ability of cells to repair this damage is thought to provide a degree of natural protection. To investigate the repair process, we have damaged plasmids containing the firefly luciferase gene with either SM or its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES). Damaged plasmids were transfected into wild-type and nucleotide excision repair (NER) deficient Chinese hamster ovary cells; these cells were also transfected with a second reporter plasmid containing RENILLA: luciferase as an internal control on the efficiency of transfection. Transfected cells were incubated at 37 degrees C for 27 h and then both firefly and RENILLA: luciferase intensities were measured on the same samples with the dual luciferase reporter assay. Bioluminescence in lysates from cells transfected with damaged plasmid, expressed as a percentage of the bioluminescence from cells transfected with undamaged plasmid, is increased by host cell repair activity. The results show that NER-competent cells have a higher reactivation capacity than NER-deficient cells for plasmids damaged by either SM or CEES. Significantly, NER-competent cells are also more resistant to the toxic effects of SM and CEES, indicating that NER is not only proficient in repairing DNA damage caused by either agent but also in decreasing their toxicity. This host cell repair assay can now be used to determine what other cellular mechanisms protect cells from mustard toxicity and under what conditions these mechanisms are most effective.

Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences

“…HR was reported to be the major repair pathway protecting cells against acute SM and HN2 toxicity, with NER and NHEJ also contributing to cell survival (Inturi et al, 2014;Jowsey et al, 2010Jowsey et al, , 2012Moller et al, 2000;Muller et al, 2000). In contrast, repair of CEES induced DNA monoadducts seem to rely mostly on BER and NER pathways (Jowsey et al, 2009;Matijasevic et al, 2001). Thus, even though mustards do not induce DNA strand breaks directly, strand breaks can occur in response to mustard treatment by enzymatic processes that actively introduce them in the course of DNA repair processes.…”

Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions

“…Alternatively, different PARy lation dynamics may be a direct consequences of the different types of DNA lesions formed, i.e., H 2 O 2 induces mainly oxidative damage and strand breaks, while CEES induces mainly DNA alkylation, without being thought to induce DNA strand breaks directly. Previous reports showed that CEES induced DNA mono adducts are predominantly repaired by BER and NER pathways (Jowsey et al, 2009;Matijasevic et al, 2001). PARylation plays an active role in both repair pathway, potentially by being activated through DNA strand breaks introduced by endonucleases as intermediates during the repair processes (Fischer et al, 2014;Pines et al, 2013Pines et al, , 2012Robert et al, 2013).…”

Versatile functions of p53 protein in multicellular organisms

“…Several characteristic conditions have been described that lead to activation of p53, which include the exhaustion of nucleotide pools [109], faults in cytoskeleton (altered polymerization of actin fibers and microtubules) [110][111][112], altered ribosome biogenesis [113], the condition of hypoxia and ischemia [14], hyperoxia [115], lack or deficit of certain growth factors and cytokines [116,117], altered adhesion and focal contacts [118], defective integrins [119], abnormal attachment of cells to a substrate (leading to the p53-dependent anoikis, or death of unattached cells) [120], accumulation of polyploid cells [32,121], formation of micronuclei [122], destruction or malformation of mitotic spindle [112], hypo-and hyperthermia [123,124], and exposure to nitric oxide (NO) [125]. These conditions induce characteristic modifications, either within the protein molecule of p53, or in the systems that control levels and activity of p53.…”