Quantitation of DNA adducts by stable isotope dilution mass spectrometry

Abstract

Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations and potentially contributing to the development of cancer. Because of their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples, including immunoassay, HPLC, and ³²P-postlabeling, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adduct concentrations in biological samples are between 0.01-10 adducts per 10⁸ normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry, especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS, have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke.

Figure 1

Ion formation in electrospray ionization mass spectrometry. Sample dissolved in an aqueous solvent is continuously sprayed through a stainless narrow bore capillary maintained under a high voltage relative to the ion sampling orifice (3–4 kV), leading to the formation of a mist of fine, highly charged droplets. The solvent is removed using heat and/or drying gas, generating either protonated or deprotonated analyte ions in the gas phase.

Figure 2

Schematic of a quandrupole mass analyzer. Four precisely engineered, parallel rods are arranged symmetrically in a square array, with opposite pairs of electrodes electrically connected. A quadrupole field is created by applying a positive direct current (dc) at a potential U with a superimposing radiofrequency (rf) potential V cosωt to the first electrode pair. The alternating pair of electrodes receives the dc potential of – U and an rf potential of V cosωt, which is out of phase by 180°. At a given value of U and V, only ions of specified mass to charge ratio (m/z) have stable trajectories and are able to move through the quadrupole mass filter and reach the detector, while all the other ions are lost.

Figure 3

Schematic of a triple quadrupole mass analyzer (Q₁q₂Q₃). Triple quadrupole instruments contain two quadrupole mass filters (Q₁ and Q₃) separated by a quadrupole collision cell/ion guide (q₂). IN a typical experiment, analyte ions (precursor ions) are selected in the first mass analyzer (Q₁) and then are accelerated into the second quadrupole (q₂) utilized as a collision cell. Analyte ions selected in Q₁ are fragmented in q₂ to produce mass fragments, which are analyzed in Q₃.

Figure 4

QqTOF hybrid mass analyzers couple a mass selective quadrupole and a collision cell to a TOF analyzer. The QqTOF design is similar to a triple quadrupole (Figure 3), with the exception that the third quadrupole is replaced by a TOF mass analyzer. The QqTOF can be operated in SIM/SRM scanning mode similar to that of a triple quadripole, but afford an improved mass resolution for the fragment ions, allowing to increased selectivity and improved signal to noise ratios.

Figure 5

Drawing of an Orbitrap mass analyzer: (a) transfer octapole; (b) curved RF-only quadrupole (C-trap); (c) octapole collision cell; (d) gate electrode; (e) trap electrode; (f) inner orbitrap electrode (central electrode); (g) outer orbitrap electrode.

Figure 6

Validation results for column switching HPLC-ESI⁺-MS/MS analysis of 1,N⁶-(2- hydroxy-3-hydroxymethylpropan-1,3-diyl)-dA in rodent DNA. Reprinted with permission from Goggin, M., Seneviratne, U., Swenberg, J. A., Walker, V. E., and Tretyakova, N. (2010) Column switching HPLC-ESI⁺-MS/MS methods for quantitative analysis of exocyclic dA adducts in the DNA of laboratory animals exposed to 1,3-butadiene. Chem. Res. Toxicol. 23, 808–812. Copyright 2010 American Chemical Society.

Figure 7

NanoLC-nanoESI⁺-MS/MS analysis of N7G-N⁶A-BD in liver DNA from a mouse exposed to 625 ppm BD for 2 weeks. Reprinted with permission from Goggin, M., Sangaraju, D., Walker, V. E., Wickliffe, J., Swenberg, J. A., and Tretyakova, N. (2011) Persistence and repair of bifunctional DNA adducts in tissues of laboratory animals exposed to 1,3-butadiene by inhalation. Chem. Res. Toxicol. 24, 809–817. Copyright 2011 American Chemical Society.

Figure 8

Diagram of valve positions employed in column switching HPLC-ESI-MS/MS. Sample is loaded onto trapping column in position A and washed to remove contaminants. In position B, the second pump backflushes the analyte from the trapping column onto the analytical column and into the mass spectrometer.

Figure 9

Column switching capLC-ESI⁺-MS/MS analysis of 1,N⁶-HMHP-dA in liver DNA from a mouse exposed to 200 ppm BD for 2 weeks. Reprinted with permission from Goggin, M., Seneviratne, U., Swenberg, J. A., Walker, V. E., and Tretyakova, N. (2010) Column switching HPLC-ESI⁺-MS/MS methods for quantitative analysis of exocyclic dA adducts in the DNA of laboratory animals exposed to 1,3-butadiene. Chem. Res. Toxicol. 23, 808–812. Copyright 2010 American Chemical Society.

Figure 10

Capillary HPLC- accurate mass nanospray MS/MS analysis of N7-trihydroxybutylguanine adducts of 1,3-butadiene in DNA extracted from blood of a confirmed smoker. Two separate mass transitions are used to monitor the analyte and the internal standard (¹⁵N₅- THBG).

Scheme 1

Role of DNA adducts in chemical carcinogenesis.

Scheme 2

Nucleobase positions in DNA frequently modified by electrophiles.

Scheme 3

The mechanism of G to A transition mutations induced by O⁶-alkylguanines.

Scheme 4

Sample processing scheme for isotope dilution HPLC-ESI-MS/MS analysis of DNA adducts.

Scheme 5

Mass spectrometry scanning modes.

Chart 1

Structures of representative DNA adducts formed from natural products and drugs: N7- melphalan-deoxyguanosine (mel-dGuo); N,N-bis[2-(N7-guaninyl) ethyl] amine DNA-DNA cross-links (G-NOR-G); cisplatin 1,2-guanine-guanine intrastrand cross-link (CP-d(GpG); 4- hydroxyestradiol-N7-guanine (4-OHE₂-N₇G); 7-(deoxyadenosin-N⁶-yl)aristolactam I (dA-AAI); (E)-α-(deoxyguanosin-N²-yl) tamoxifen (dG-Tam); (E)-α-(deoxyguanosin-N²-yl)-N-desmethyl tamoxifen (dG-desMeTam); aflatoxinB¹- N7-guanine (AFB₁- N7-Gua), 17α-ethynylestradiol-2′-deoxyadenosine; estrone-2′-deoxyguanosine, equilenin-2′-deoxyguanosine; estradiol-2′-deoxyguanosine.

Chart 2

Structures of DNA adducts formed by aromatic amines, N-(deoxyguanosin-8-yl)-4- ABP (C8- dG-4-ABP); N-(deoxyguanosine-8-yl)-2-amino-3-methylimidazo [4,5-f] quinoline (C8-dG-IQ); N-(deoxyguanosine-8-yl)-MeIQx (C8- dG-MeIQ); 5-(deoxyguanosin-N²-yl)-MeIQx (N²-dG- MeIQ); and 5-(deoxyguanosine-N²-yl)-2-amino-3-methylimidazo [4,5-f] quinoline (N²- dG-IQ).

Chart 3

Nitrosamine-derived DNA adducts: glyoxal-deoxyguanosine (gdG); O⁶-2- hydroxyethyl-2′-doexyguanosine (OHEdG); O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (O⁶-POB-dG); O⁶-[4-(3-pyridyl)-4-hydroxybut-1-yl]-2′-deoxyguanosine (O⁶-PHB-dG); 7-[4-(3- pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua); 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]guanine (7- PHB-Gua); O²-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O²-POB-Cyt); O²-[4-(3-pyridyl)-4- ohydroxybut-1-yl]cytosine (O²-PHB-Cyt); O²-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O²-POBdThd); O²-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (O²-POB-dThd); O⁶-methyl-2′-deoxyguanosine (O⁶-Me-dG); N7-methylguanine (N7-Me-G); O⁶-ethyl-2′-deoxyguanosine (O⁶- Et-dG).

Chart 4

Exocyclic DNA adduct structures, 1,N⁶-etheno-adenine (εA); 1,N⁶-etheno-2′-deoxyadenosine (εdA); 3,N⁴-etheno-2′-deoxycytosine (εdC); 1,N²-etheno-2′-deoxyguanosine (εdG); hexenal-derived exocyclic 1, N²-propanodeoxyguanosine (Hex-PdG); glyoxal-2′-deoxyguanosine (gdG); 1, N²-propanodeoxyguanosine; acrolein-dG (Acro-dG); 1,N²- hydroxynonenal-dG (HNE-dG); and N²-(1-carboxyethyl)-2′-deoxyguanosine (CEdG).

Chart 5

Structures of ethanol and acetaldehyde induced DNA adducts: N²-ethylidene-2′-deoxyguanosine (N²-ethylidene-dG); N²-ethyl-2′-deoxyguanosine (N²-ethyl-dG); C8-(1- hydroxyethyl)guanine (C8-(1-HE)-Gua; and N7-ethylguanine (N7-ethyl-G).

Chart 6

Polycyclic aromatic hydrocarbon-derived DNA adducts: 7,8,9-trihydroxy-10-( N²- deoxyguanosyl)-7,8,9,10-tretrahydrobenzo[a]pyrene (N²-BPDE-dG); N7-(benzo[a]pyrene-6- yl)guanine (BP-6-N7Gua); methypyrene-2′-deoxyadenosine (MP-dAdo); and methylpyrene-2′-deoxyguanosine (MP-dGuo).

Chart 7

Structures of epoxide induced DNA adducts, N7-(2,3,4-trihydroxybutyl)guanine (N7- THBG); 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD); 1-(guan-7-yl)-4-(aden-1-yl)-2,3- butanediol (N7G-N1A-BD); 1,N⁶-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (1,N⁶ -αHMHP-dA); 1,N⁶-(2-hydroxy-3-hydroxymethyl-propan-1,3-diyl)-2′-deoxyadenosine (1,N⁶ -γHMHP-dA); N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua); and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade).

Chart 8

Structures of oxidative DNA adducts, 8-oxo-7,8-dihydro-2′deoxyguanosine (8-oxo-dG); 8-oxo-7,8-dihydro-2′deoxyadenosine (8-oxo-dA); 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl) amino]-5(2H)-oxazolone (oxazolone).