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

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 ultra-performance liquid chromatography-electrospray ionization/multistage mass spectrometry (UPLC-ESI/MSn) with a linear quadrupole ion-trap mass spectrometer, to measure 7-(deoxyadenosin-N6-yl) aristolactam I (dA-AL-I) and 7-(deoxyguanosin-N2-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 108 DNA bases. We have used UPLC-ESI/MSn 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 108 DNA bases, whereas dG-AL-I was not found. We conclude that UPLC-ESI/MSn is a highly sensitive, specific and robust analytical method, positioned to supplant 32P-postlabeling techniques currently used for biomonitoring of DNA adducts in human tissues. Importantly, UPLC-ESI/MSn could be used to document exposure to AA, the toxicant responsible for AA nephropathy and its associated UUC.

New approaches for biomonitoring exposure to the human carcinogen aristolochic acid

“…21 Even though the amounts of AA-II present in Aristolochia herbs are usually within two- to five-fold of the amounts of AA-I, 19,70,71 the dA-AL-II adduct was detected at a frequency of only several percent and occurred at levels ~100-fold lower than the levels of dA-AL-I. 21,38,51 This DNA adduct biomarker data implicate dA-AL-I as the major genotoxic lesion in humans.…”

“…38 Subsequently, stable isotopically labelled [ 15 N 5 ]-dA-AL-I and [ 15 N 3 ]-dA-AL-II were synthesized and employed for quantitative mass spectrometric measurements of these adducts. 51 Representative mass chromatograms and product ion spectra of dA-AL-I measured in the renal cortex of three patients with upper urothelial cancer from Taiwan, by UPLC-ESI/MS 3 , are shown in Figure 4. The level of adducts in the positive samples were 0.4 adducts and 5.9 adducts per 10 8 bases.…”

“…69 Of the 148 Taiwanese subjects assayed by MS, 132 subjects harbored dA-AL-I at a level above 0.3 adducts per 10 8 DNA bases (the LOQ value) in the renal cortex (unpublished data). 25,45,51 The mean level of dA-AL-I was 16.0 adducts per 10 8 DNA bases with a 95% confidence interval range from 10.1 to 21.0 adducts per 10 8 DNA bases (a value of 0.15 adducts per 10 8 DNA bases, one-half the LOQ value for non-positive samples, was used for the calculation of the 95% CI).…”

Low-Coverage Exome Sequencing Screen in Formalin-Fixed Paraffin-Embedded Tumors Reveals Evidence of Exposure to Carcinogenic Aristolochic Acid

“…AL-DNA adducts may persist for over 20 years after the exposure had ceased (15, 16) and can be measured by 32 P-postlabelling (14, 17) or by ultra-performance-liquid chromatography-electrospray ionization-multistage scan mass spectrometry (UPLC-ESI-MS/MS n ), both applicable to formalin-fixed paraffin-embedded (FFPE) tissues (16, 18, 19). However, the 32 P-postlabeling method lacks specificity, and access to the UPLC-ESI-MS/MS n methodology and its optimization for biomaterial of low quantity are limiting factors.…”

Application of liquid chromatography-mass spectrometry and differential mobility spectrometry-mass spectrometry for detection and quantitation of biomarkers in complex biological matrices

“…Another important effect of kidney damage is the presence of albumin or other protein in the urine. This measurement of proteinuria is definitive but is somewhat lacking in sensitivity, so combinations of protein tests and urine creatinine are also used for assessing kidney function [99].…”