Application of DNA adductomics to soil bacterium Sphingobium sp. strain KK22

Abstract

Toward the development of ecotoxicology methods to investigate microbial markers of impacts of hydrocarbon processing activities, DNA adductomic analyses were conducted on a sphingomonad soil bacterium. From growing cells that were exposed or unexposed to acrolein, a commonly used biocide in hydraulic fracturing processes, DNA was extracted, digested to 2'-deoxynucleosides and analyzed by liquid chromatography-positive ionization electrospray-tandem mass spectrometry in selected reaction monitoring mode transmitting the [M + H](+) > [M + H - 116](+) transition over 100 transitions. Overall data shown as DNA adductome maps revealed numerous putative DNA adducts under both conditions with some occurring specifically for each condition. Adductomic analyses of triplicate samples indicated that elevated levels of some targeted putative adducts occurred in exposed cells. Two exposure-specific adducts were identified in exposed cells as 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxy-(and 8-hydroxy-)pyrimido[1,2-a]- purine-(3H)-one (6- and 8-hydroxy-PdG) following synthesis of authentic standards of these compounds and subsequent analyses. A time course experiment showed that 6- and 8-hydroxy-PdG were detected in bacterial DNA within 30 min of acrolein exposure but were not detected in unexposed cells. This work demonstrated the first application of DNA adductomics to examine DNA damage in a bacterium and sets a foundation for future work.

Keywords: Acrolein; DNA adductomics; DNA adducts; LC-MS/MS; Sphingobium; bacterium.

Figure 1

Adductome analyses of bacterial cells growing on glucose in mid-log phase (A), and bacterial cells growing on glucose in mid-log phase 30 min after exposure to acrolein (B). Putative DNA adducts discussed in the text are labeled I through VII and a through d.

Figure 2

LC/ESI(+)-MS/MS product ion scan analyses of putative DNA adduct III (10 eV) which corresponded to the protonated molecule [M + H]⁺ = 312 and which was revealed by DNA adductome analysis in Figure1. Inset, results of CID fragmentation at 20 eV allowed for the detection of the m/z 117 deoxyribose diagnostic fragment. LC/ESI (+)-MS/MS, liquid chromatography electrospray ionization-tandem mass spectrometry in positive ionization mode; CID, collision induced dissociation.

Figure 3

(A) LC separation of a mixture of authentic standards of 2′-deoxynucleosides 2′-deoxycytidine, 2′-deoxythymidine, 2′-deoxyguanosine, 2′-deoxyadenosine and N⁶-methyl-2′-deoxyadenosine plus dideoxyinosine (dC, dG, dT, dA, N⁶-medA and ddI respectively). (B) N⁶-methyl-2′-deoxyadenosine detected by LC/ESI(+)-MS/MS SRM mode at transition 266 > 150, and (C) putative DNA adduct VII detected by LC/ESI(+)-MS/MS SRM mode at transition 266 > 150. The peak that was detected at 9.75 min occurred due to the abundantly detected sodiated ion of 2′-deoxythymidine which was detected at SRM transition 265 > 149. LC/ESI (+)-MS/MS, liquid chromatography electrospray ionization-tandem mass spectrometry in positive ionization mode; SRM, single reaction monitoring.

Figure 4

Results of LC/ESI(+)-MS/MS product ion scan analyses of (A) putative DNA adduct c, which corresponded to the protonated molecule [M + H]⁺ = 324, (B) an authentic standard of 8-hydroxy-PdG, and (C) putative DNA adduct d, which corresponded to the protonated molecule [M + H]⁺ = 346. Analyses were conducted at a CID of 20 eV. [BH₂]⁺ is used to represent the doubly protonated base ion, [M + 2H − dR]⁺. LC/ESI (+)-MS/MS, liquid chromatography electrospray ionisation-tandem mass spectrometry in positive ionisation mode; CID, collision induced dissociation.

Figure 5

Results of LC/ESI(+)-MS/MS SRM mode time course analyses of DNA-derived 2′-deoxynucleosides extracted from a soil bacterium. SRM transition 324 > 208 was monitored in samples taken at T = 0 (A and B), T = 8 (C and D) and T = 30 min (E and F) from cells growing on glucose (g) and from cells growing on glucose after exposure to acrolein (g + a). The signal to noise (S/N) ratios for putative DNA adduct peaks at 11.7 and 12.0 min in (F) were six and 10 respectively. The results of analysis of three authentic standards of 6-, 6-, and 8-hydroxy-PdG which eluted at retention times of 11.2, 11.7, and 12.0 min, respectively, are given in (G). LC/ESI (+)-MS/MS, liquid chromatography electrospray ionization-tandem mass spectrometry in positive ionization mode; SRM, single reaction monitoring.

Figure 6

Results of quantification of 8-hydroxy-PdG in the DNA of bacterial cells that were exposed or not exposed to acrolein.