Effects of peroxynitrite dose and dose rate on DNA damage and mutation in the supF shuttle vector

Abstract

Peroxynitrite (ONOO-) induces oxidative and nitrosative DNA damage, and previous studies by our group have shown that it is strongly mutagenic in the supF shuttle vector pSP189 replicated in Escherichia coli MBL50 cells. In those experiments, however, the pSP189 plasmid was exposed under unphysiological conditions to large single bolus doses of ONOO-, which limits extrapolation of the data to in vivo pathological states in which ONOO- may play a role. We have thus sought to define the effects of ONOO- dose and dose rate on the DNA damage and mutations induced in the supF gene by three different dosage mechanisms: (i) by infusion of ONOO- solution into suspensions of pSP189 at rates approximating those estimated to occur in inflamed tissues; (ii) by exposure to 3-morpholinosydnonimine (SIN-1), which generates ONOO- spontaneously during decomposition; and (iii) by bolus doses of ONOO- solution. In all cases, plasmid DNA was exposed in the presence of 25 mM bicarbonate, since the reaction of CO2 with ONOO- (to form nitrosoperoxycarbonate) has a major impact on mutagenic potency of ONOO- in this system. Nucleobase and deoxyribose damage were evaluated by a plasmid nicking assay immediately after ONOO- and SIN-1 exposures. Mutation frequency (MF) and mutational spectra in the supF gene were determined after plasmid pSP189 replicated in host E. coli cells. Bolus ONOO- addition caused the highest amount of DNA damage, including base and deoxyribose lesions, while infusion caused the least. SIN-1 was found to induce almost exclusively deoxyribose oxidation, while bolus addition generated a high percentage of base damage. MF increased in a dose-dependent manner following all treatments, but infused ONOO- and SIN-1 exposures were less mutagenic than bolus ONOO- exposure. MFs induced by infusion and by SIN-1 incubated for 100 min at the highest level (4 mM) were 63 and 43% less, respectively, than that induced by bolus. All mutational hot spots were located at G:C sites except for A121 and A177 induced by SIN-1 exposure. Hot spots at C108 and C168 were common to all exposures; G113, G115, and G116 were common to bolus and infused ONOO- exposures; and G129 was common to infused ONOO- and SIN-1 exposures. Almost all mutations were single base pair substitutions under all exposure conditions. Whereas those induced by infused or bolus ONOO- and SIN-1 consisted predominantly of G:C to T:A transversions (66, 65, and 51%, respectively), G:C to C:G mutations were much less frequent following infusion and SIN-1 (8 and 19%, respectively) than those induced by bolus exposure (29%). A:T to T:A mutations induced were detected only after ONOO- infusion and SIN-1 exposure (9 and 11%, respectively). In conclusion, both dose and dose rate at which a genetic target is exposed to ONOO- substantially influence the damage and mutational response, indicating that these parameters will need to be taken into account in assessing the potential effects of ONOO- in vivo. Furthermore, the results indicate that the chemistry of SIN-1-induced DNA damage differs substantially from native ONOO-, which suggests the need for caution in interpreting the biological relevance of SIN-1 as a surrogate for ONOO-.