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. 2012 May 21;25(5):1119-31.
doi: 10.1021/tx3000889. Epub 2012 May 4.

Biomonitoring of aristolactam-DNA Adducts in Human Tissues Using Ultra-Performance Liquid Chromatography/Ion-Trap Mass Spectrometry

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Biomonitoring of aristolactam-DNA Adducts in Human Tissues Using Ultra-Performance Liquid Chromatography/Ion-Trap Mass Spectrometry

Byeong Hwa Yun et al. Chem Res Toxicol. .
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Abstract

Aristolochic acids (AAs) are a structurally related family of nephrotoxic and carcinogenic nitrophenanthrene compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs have been implicated in the etiology of so-called Chinese herbs nephropathy and of Balkan endemic nephropathy. Both of these disease syndromes are associated with carcinomas of the upper urinary tract (UUC). 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I) is a principal component of Aristolochia herbs. Following metabolic activation, AA-I reacts with DNA to form aristolactam (AL-I)-DNA adducts. We have developed a sensitive analytical method, using ultraperformance liquid chromatography-electrospray ionization/multistage mass spectrometry (UPLC-ESI/MS(n)) with a linear quadrupole ion-trap mass spectrometer, to measure 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I) and 7-(deoxyguanosin-N(2)-yl) aristolactam I (dG-AL-I) adducts. Using 10 μg of DNA for measurements, the lower limits of quantitation of dA-AL-I and dG-AL-I are, respectively, 0.3 and 1.0 adducts per 10(8) DNA bases. We have used UPLC-ESI/MS(n) to quantify AL-DNA adducts in tissues of rodents exposed to AA and in the renal cortex of patients with UUC who reside in Taiwan, where the incidence of this uncommon cancer is the highest reported for any country in the world. In human tissues, dA-AL-I was detected at levels ranging from 9 to 338 adducts per 10(8) DNA bases, whereas dG-AL-I was not found. We conclude that UPLC-ESI/MS(n) is a highly sensitive, specific and robust analytical method, positioned to supplant (32)P-postlabeling techniques currently used for biomonitoring of DNA adducts in human tissues. Importantly, UPLC-ESI/MS(n) could be used to document exposure to AA, the toxicant responsible for AA nephropathy and its associated UUC.

Figures

Figure 1
Figure 1
Metabolic activation of AA and its reaction with DNA. Adduct formation occurs after reduction of the nitro moiety of the phenanthrene rings of AA-I and AA-II to form the N-hydroxyaristolactams and proposed nitrenium ions, which react with dG and dA to form dA-AL-I, dG-AL-I, dA-AL-II, and dG-AL-II.
Figure 2
Figure 2
Product ion spectra of AL-DNA adducts acquired at the MS3 scan stage, representing the fragmentation of the aglycone adducts [BH2]+ of dG-AL-II (m/z 413), dG-AL-I (m/z 443), dA-AL-II (m/z 397), and dA-AL-I (m/z 427). Based upon the comparison of the product ion spectra of the dA-AL-II and [15N3]-dA-AL-II adducts (Supporting Information, Figure S5), the fragment ion at m/z 324.2 (m/z 327.2 for [15N3]-dA-AL-II) may form by the loss of CH2O and HNCO from the substituted phenanthrene ring.
Figure 3
Figure 3
The mass chromatograms of the AL-modified genomic DNA from a mouse dosed with AA-I (1 mg/kg). Mouse liver and kidney DNA were diluted with unmodified CT DNA by a factor of 10 and 5, respectively. The level of 15N-labeled internal standards was 5 adducts per 108 bases. The levels of AL-DNA adducts expressed per 108 bases in undiluted mouse liver and kidney were dG-AL-I: 58.1 (liver) and 300 (kidney); dA-AL-II: 0.7 (liver) and 0.8 (kidney); dA-AL-I: 176 (liver) and 1017 (kidney). The combined ions presented in the mass chromatograms of the adducts and internal standards were employed for quantitative measurements. The trace amounts of AL-DNA adducts detected in untreated DNA are attributed to the residual unlabeled dG and dA present in the isotopically labelled internal standards.
Figure 4
Figure 4
The mass chromatograms of dG-AL-I and dA-AL-I adducts in mouse kidney DNA diluted with unmodified CT DNA to levels approaching the LOQ. Mass chromatograms of untreated and undiluted mouse liver DNA (left panel); AA-I treated mouse kidney (right panel) with dG-AL-I estimated at 1.3 adduct per 108 bases and dA-AL-I estimated at 0.3 adducts per 108 bases. The internal standards were added at a level of 5.0 adducts per 108 bases.
Figure 5
Figure 5
32P-postlabeling/PAGE analysis of AL-DNA adducts. DNA was obtained from the renal cortex of a mouse treated with AA-I (2 mg/kg) and diluted with unmodified DNA (20 μg total) to arrive at levels of modification of 3, 7, 21 and 108 dA-AL-I adducts per 109 bases. For this experiment, the levels of dG-AL-I adducts are 3 times less than dA-AL-I. The experiment was performed in quadruplicate for the lowest adduct level and in triplicate for higher levels. (A) Lanes 1 and 15 (dA-AL-II and dG-AL-II standards, 80 adducts/109 bases); lanes 2–4 (100 dA-AL-I adducts per 109 bases); lanes 5–7 (21 dA-AL-I adducts per 109 bases); lanes 8–10 (7 dA-AL-I adducts per 109 bases); lanes 11–14 (3 dA-AL-I adducts per 109 bases). Upper and lower bands correspond to dG-AL and dA-AL adducts, respectively. (B) Dose response for dA-AL-I and (C) dose response for dG-AL-I levels measured with Image QuaNT v5.2 software were plotted as a function of adduct modification.
Figure 6
Figure 6
The mass chromatograms of dG-AL-I and dA-AL-I in renal cortex DNA from two patients with UUC. (A) unmodified CT DNA, (B) Subject C95, (C) Subject C112. The human DNA was diluted with unmodified CT DNA by a factor of 6 for subject 1 and and 10-fold for subject 2. The estimated levels of dA-AL-I were 142 adducts per 108 bases for subject 1 and 44.8 adducts per 108 bases for subject 2 in non-diluted DNA.
Figure 7
Figure 7
The mass chromatograms of dA-AL-I adduct in unmodified CT DNA and DNA from human UUC tissue. Human DNA (5 μg) was diluted with CT DNA (15 μg). The dA-AL-I adduct was estimated at 0.9 adducts per 108 DNA bases in the undiluted sample.

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