Molecular profiles and urinary biomarkers of upper tract urothelial carcinomas associated with aristolochic acid exposure

Recurrent upper tract urothelial carcinomas (UTUCs) arise in the context of nephropathy linked to exposure to the herbal carcinogen aristolochic acid (AA). Here we delineated the molecular programs underlying UTUC tumorigenesis in patients from endemic aristolochic acid nephropathy (AAN) regions in Southern Europe. We applied an integrative multiomics analysis of UTUCs, corresponding unaffected tissues and of patient urines. Quantitative microRNA (miRNA) and messenger ribonucleic acid (mRNA) expression profiling, immunohistochemical analysis by tissue microarrays and exome and transcriptome sequencing were performed in UTUC and nontumor tissues. Urinary miRNAs of cases undergoing surgery were profiled before and after tumor resection. Ribonucleic acid (RNA) and protein levels were analyzed using appropriate statistical tests and trend assessment. Dedicated bioinformatic tools were used for analysis of pathways, mutational signatures and result visualization. The results delineate UTUC-specific miRNA:mRNA networks comprising 89 miRNAs associated with 1,862 target mRNAs, involving deregulation of cell cycle, deoxyribonucleic acid (DNA) damage response, DNA repair, bladder cancer, oncogenes, tumor suppressors, chromatin structure regulators and developmental signaling pathways. Key UTUC-specific transcripts were confirmed at the protein level. Exome and transcriptome sequencing of UTUCs revealed AA-specific mutational signature SBS22, with 68% to 76% AA-specific, deleterious mutations propagated at the transcript level, a possible basis for neoantigen formation and immunotherapy targeting. We next identified a signature of UTUC-specific miRNAs consistently more abundant in the patients' urine prior to tumor resection, thereby defining biomarkers of tumor presence. The complex gene regulation programs of AAN-associated UTUC tumors involve regulatory miRNAs prospectively applicable to noninvasive urine-based screening of AAN patients for cancer presence and recurrence

Physiological and Molecular Characterization of Aristolochic Acid Transport by the Kidney[S]

Consumption of herbal medicines derived from Aristolochia plants is associated with a progressive tubulointerstitial disease known as aristolochic acid (AA) nephropathy. The nephrotoxin produced naturally by these plants is AA-I, a nitrophenanthrene carboxylic acid that selectively targets the proximal tubule. This nephron segment is prone to toxic injury because of its role in secretory elimination of drugs and other xenobiotics. Here, we characterize the handling of AA-I by membrane transporters involved in renal organic anion transport. Uptake assays in heterologous expression systems identified murine organic anion transporters (mOat1, mOat2, and mOat3) as capable of mediating transport of AA-I. Kinetic analyses showed that all three transporters have an affinity for AA-I in the submicromolar range and thus are likely to operate at toxicologically relevant concentrations in vivo. Structure-activity relationships revealed that the carboxyl group is critical for high-affinity interaction of AA-I with mOat1, mOat2, and mOat3, whereas the nitro group is required only by mOat1. Furthermore, the 8-methoxy group, although essential for toxicity, was not requisite for transport. Mouse renal cortical slices avidly accumulated AA-I, achieving slice-to-medium concentration ratios \u3e10. Uptake by slices was sensitive to known mOat1 and mOat3 substrates and the organic anion transport inhibitor probenecid, which also blocked the production of DNA adducts formed with reactive intracellular metabolites of AA-I. Taken together, these findings indicate that OAT family members mediate high-affinity transport of AA-I and may be involved in the site-selective toxicity and renal elimination of this nephrotoxin

Cytochrome P450 1A2 Detoxicates Aristolochic Acid in the Mouse

Aristolochic acids (AAs) are plant-derived nephrotoxins and carcinogens responsible for chronic renal failure and associated urothelial cell cancers in several clinical syndromes known collectively as aristolochic acid nephropathy (AAN). Mice provide a useful model for study of AAN because the renal histopathology of AA-treated mice is strikingly similar to that of humans. AA is also a potent carcinogen in mice with a tissue spectrum somewhat different from that in humans. The toxic dose of AA in mice is higher than that in humans; this difference in susceptibility has been postulated to reflect differing rates of detoxication between the species. Recent studies in mice have shown that the hepatic cytochrome P450 system detoxicates AA, and inducers of the arylhydrocarbon response protect mice from the nephrotoxic effects of AA. The purpose of this study was to determine the role of specific cytochrome P450 (P450) enzymes in AA metabolism in vivo. Of 18 human P450 enzymes we surveyed only two, CYP1A1 and CYP1A2, which were effective in demethylating 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI) to the nontoxic derivative 8-hydroxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAIa). Kinetic analysis revealed similar efficiencies of formation of AAIa by human and rat CYP1A2. We also report here that CYP1A2-deficient mice display increased sensitivity to the nephrotoxic effects of AAI. Furthermore, Cyp1a2 knockout mice accumulate AAI-derived DNA adducts in the kidney at a higher rate than control mice. Differences in bioavailability or hepatic metabolism of AAI, expression of CYP1A2, or efficiency of a competing nitroreduction pathway in vivo may explain the apparent differences between human and rodent sensitivity to AAI

Variation in Presentation and Presence of DNA Adducts and p53 Mutations in Patients with Endemic Nephropathy - an Environmental Form of the Aristolochic Acid Nephropathy

Background: Endemic nephropathy (EN) and associated urothelial cell cancers (UUC) are an environmental form of aristolochic acid nephropathy where the most probable rout of ingestion of aristolochic acid (AA) was made by bread contaminated with AA, leading to chronic dietary intoxication. Clinical courses of three members of the same family, similarly exposed to toxin, who exhibited different clinical courses of the disease are presented. Methods: Questionnaires on AA exposure were taken. Tissue samples were obtained during therapeutic nephrouretectomies. Histopathology, immunohistochemical detection of p53, p53 mutation screening in tumor DNA and analysis on the presence of aristolactam (AL)-DNA adducts were performed. Results: Case 1 had UUC with typical EN histopathological signs, whereas Case 2 had bilateral UUCs with typical EN histopathological signs. In contrast, the patient in Case 3 initially showed renal insufficiency, complicated afterwards by right UUC, and later on by left UUC with histopathological end-stage chronic changes but without typical EN changes. AA-DNA adducts and specific p53 mutational spectra (A:T→ T:A transversion) were found in tissues of cases 1 and 2. Conclusion: Diverse clinical courses seem to be related not to differences in exposure but to differences in metabolic activation or detoxification of AA and/or DNA repair resulting from different genetic polymorphisms

Aristolactam-DNA adducts are a biomarker of environmental exposure to aristolochic acid

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by 32P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract

Aristolochic Acid I Metabolism in the Isolated Perfused Rat Kidney

Aristolochic acids are natural nitro-compounds found globally in the plant genus <i>Aristolochia</i> that have been implicated in the severe illness in humans termed aristolochic acid nephropathy (AAN). Aristolochic acids undergo nitroreduction, among other metabolic reactions, and active intermediates arise that are carcinogenic. Previous experiments with rats showed that aristolochic acid I (AA-I), after oral administration or injection, is subjected to detoxication reactions to give aristolochic acid Ia, aristolactam Ia, aristolactam I, and their glucuronide and sulfate conjugates that can be found in urine and feces. Results obtained with whole rats do not clearly define the role of liver and kidney in such metabolic transformation. In this study, in order to determine the specific role of the kidney on the renal disposition of AA-I and to study the biotransformations suffered by AA-I in this organ, isolated kidneys of rats were perfused with AA-I. AA-I and metabolite concentrations were determined in perfusates and urine using HPLC procedures. The isolated perfused rat kidney model showed that AA-I distributes rapidly and extensively in kidney tissues by uptake from the peritubular capillaries and the tubules. It was also established that the kidney is able to metabolize AA-I into aristolochic acid Ia, aristolochic acid Ia <i>O</i>-sulfate, aristolactam Ia, aristolactam I, and aristolactam Ia <i>O</i>-glucuronide. Rapid demethylation and sulfation of AA-I in the kidney generate aristolochic acid Ia and its sulfate conjugate that are voided to the urine. Reduction reactions to give the aristolactam metabolites occur to a slower rate. Renal clearances showed that filtered AA-I is reabsorbed at the tubules, whereas the metabolites are secreted. The unconjugated metabolites produced in the renal tissues are transported to both urine and perfusate, whereas the conjugated metabolites are almost exclusively secreted to the urine

Human Formalin-Fixed Paraffin-Embedded Tissues: An Untapped Specimen for Biomonitoring of Carcinogen DNA Adducts by Mass Spectrometry

DNA adducts represent internal dosimeters to measure exposure to environmental and endogenous genotoxicants. Unfortunately, in molecular epidemiologic studies, measurements of DNA adducts often are precluded by the unavailability of fresh tissue. In contrast, formalin-fixed paraffin embedded (FFPE) tissues frequently are accessible for biomarker discovery. We report here that DNA adducts of aristolochic acids (AAs) can be measured in FFPE tissues at a level of sensitivity comparable to freshly frozen tissue. AAs are nephrotoxic and carcinogenic compounds found in <i>Aristolochia</i> herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs are implicated in the etiology of aristolochic acid nephropathy and upper urinary tract carcinoma. 8-Methoxy-6-nitrophenanthro-[3,4-<i>d</i>]-1,3-dioxole-5-carboxylic acid (AA-I) is a component of <i>Aristolochia</i> herbs and a potent human urothelial carcinogen. AA-I reacts with DNA to form the aristolactam (AL-I)-DNA adduct 7-(deoxyadenosin-<i>N</i>⁶-yl) aristolactam I (dA-AL-I). We established a method to quantitatively retrieve dA-AL-I from FFPE tissue. Adducts were measured, using ultraperformance liquid chromatography/mass spectrometry, in liver and kidney tissues of mice exposed to AA-I, at doses ranging from 0.001 to 1 mg/kg body weight. dA-AL-I was then measured in 10-μm thick tissue-sections of FFPE kidney from patients with upper urinary tract cancers; the values were comparable to those observed in fresh frozen samples. The limit of quantification of dA-AL-I was 3 adducts per 10⁹ DNA bases per 2.5 μg of DNA. The ability to retrospectively analyze FFPE tissues for DNA adducts may provide clues to the origin of human cancers for which an environmental cause is suspected