High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of benzo[a]pyrene-DNA adducts.
A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-(2)H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 microg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10(8) nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3-(3)H]BPDE, the method gave a value very similar to those obtained by radiolabeling, (32)P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-(3)H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 +/- 0.04 dG-BPDE adducts/10(8) nucleotides. This value is much lower than the 102 +/- 14 total DNA adducts/10(8) nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/10(8) nucleotides, when using 100 microg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.
Beland
Frederick A
FA
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA. fbeland@nctr.fda.gov
Churchwell
Mona I
MI
Von Tungeln
Linda S
LS
Chen
Shoujun
S
Fu
Peter P
PP
Culp
Sandra J
SJ
Schoket
Bernadette
B
Gyorffy
Erika
E
Minárovits
János
J
Poirier
Miriam C
MC
Bowman
Elise D
ED
Weston
Ainsley
A
Doerge
Daniel R
DR
D000818:Animals; D001564:Benzo(a)pyrene / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003033:Coal Tar / Q000633:toxicity; D018736:DNA Adducts / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008175:Lung Neoplasms / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D018736:DNA Adducts; D007202:Indicators and Reagents; D001564:Benzo(a)pyrene; D003033:Coal Tar; D003903:Deuterium