New Approaches for Biomonitoring Exposure to the Human Carcinogen Aristolochic Acid

Abstract

Aristolochic acids (AA) are found in all Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes for centuries. AA are causal agents of the chronic kidney disease entity termed aristolochic acid nephropathy (AAN) and potent upper urinary tract carcinogens in humans. AAN and upper urinary tract cancers are endemic in rural areas of Croatia and other Balkan countries where exposure to AA occurs through the ingestion of home-baked bread contaminated with Aristolochia seeds. In Asia, exposure to AA occurs through usage of traditional Chinese medicinal herbs containing Aristolochia. Despite warnings from regulatory agencies, traditional Chinese herbs containing AA continue to be used world-wide. In this review, we highlight novel approaches to quantify exposure to AA, by analysis of aristolactam (AL) DNA adducts, employing ultraperformance liquid chromatography-electrospray ionization/multistage mass spectrometry (UPLC-ESI/MSⁿ). DNA adducts are a measure of internal exposure to AA and serve as an important end point for cross-species extrapolation of toxicity data and human risk assessment. The level of sensitivity of UPLC-ESI/MSⁿ surpasses the limits of detection of AL-DNA adducts obtained by ³²P-postlabeling techniques, the most widely employed methods for detecting putative DNA adducts in humans. AL-DNA adducts can be measured by UPLC-ESI/MS³, not only in fresh frozen renal tissue, but also in formalin-fixed, paraffin-embedded (FFPE) samples, an underutilized biospecimen for assessing chemical exposures, and in exfoliated urinary cells, a non-invasive approach. The frequent detection of AL DNA adducts in renal tissues, combined with the characteristic mutational spectrum induced by AA in TP53 and other genes provides compelling data for a role of AA in upper urothelial tract cancer.

Figure 1

Bioactivation of AA and DNA adduct formation. The nitroreduction of AA to form AL-NOH can lead to the formation of AL-DNA adducts. The reactivity of AL-NOH with DNA is significantly increased upon the sulfonation of AL-NOH.

Figure 2

dA-AL adducts induce the otherwise rare A-to-T transversion mutation in the TP53 gene of patients with AAN in the Taiwan and the Balkans. This figure was reproduced with permission from reference .

Figure 3

Polyacrylamide gel electrophoresis (PAGE) of ³²P-Postlabeled AL-DNA adducts from an American woman who developed end-stage renal failure after treatment with an herbal remedy containing Aristolochia. Samples in lanes 1–3 and 4–6 were excised from the right and leftkidneys, respectively. Lanes 1 and 4 are from the renal cortex; lanes 2 and 5, from the renal medulla; and lanes 3 and 6, from the renal pelvis. Oligonucleotides containing dA-AL-I and dG-AL-I (1 adduct per 10⁶ dNs), digested in parallel, were used as standards. The figure was reproduced with permission from reference and “Copyright (2007) National Academy of Sciences, U.S.A.”

Figure 4

UPLC-ESI/MS³ reconstructed ion chromatograms of dA-AL-I from human kidney cortex of patients with upper urothelial cancer from Taiwan at levels (A) below the LOQ, and positive samples at (B) 0.4 adducts, and (C) 5.9 adducts per 10⁸ bases. The product ion spectra of dA-AL-I from subject C is depicted along with the internal standard [¹⁵N₅]-dA-AL-I (¹⁵N labels are depicted with asterisks), which was added to DNA at a level of 5 adducts per 10⁸ bases.

Figure 5

Frequency and levels of dA-AL-I adduct identified in renal cortex of subjects from the Taiwan cohort. 132 out of the 148 subjects assayed harbored dA-AL-I at a level above 0.3 adducts per 10⁸ DNA bases (the LOQ value).

Figure 6

The mean and maximum range of dA-AL-I adduct levels in cortex tissue of subjects from Taiwan (left scale axis) and the Balkans (right scale axis).

Figure 7

dA-AL-I adduct formation in C57BL/6J mice exposed to AA-I (0.001–1 mg/kg body weight). The overall mean difference in adduct levels between freshly frozen and FFPE kidney and liver tissues, fixed in formalin for 24 hours across all doses was 21 ± 14% (mean ± SD, N = 4). This figure was reproduced with permission from reference .

Figure 8

dA-AL-I adducts in matching fresh frozen and FFPE kidney samples, containing both renal cortex and medulla, obtained from 11 individuals residing in endemic regions of Croatia and Serbia who underwent nephroureterectomy for UTUC, and a representative FFPE paraffin-embedded renal tissue block.

Figure 9

UPLC-ESI/MS³ chromatograms of dA-AL-I present in human exfoliated urinary cells. Analysis was done with 3 to 6.5 μg DNA. The internal standard, [¹⁵N₅]-dA-AL-I, was added at a level of 3.9 adducts per 10⁸ bases for subject 13; 8.3 adducts per 10⁸ bases for subject 17; and 5.6 adducts per 10⁸ bases for subject 19. The product ion spectrum of dA-AL-I and [¹⁵N₅]-dA-AL-I are shown for subject 17.