Mechanisms of antiviral action and toxicities of ipecac alkaloids: Emetine and dehydroemetine exhibit anti-coronaviral activities at non-cardiotoxic concentrations.

The emergence of highly infectious pathogens with their potential for triggering global pandemics necessitate the development of effective treatment strategies, including broad-spectrum antiviral therapies to safeguard human health. This study investigates the antiviral activity of emetine, dehydroemetine (DHE), and congeneric compounds against SARS-CoV-2 and HCoV-OC43, and evaluates their impact on the host cell. Concurrently, we assess the potential cardiotoxicity of these ipecac alkaloids. Significantly, our data reveal that emetine and the (-)-R,S isomer of 2,3-dehydroemetine (designated in this paper as DHE4) reduce viral growth at nanomolar concentrations (i.e., IC50 ∼ 50-100 nM), paralleling those required for inhibition of protein synthesis, while calcium channel blocking activity occurs at elevated concentrations (i.e., IC50 ∼ 40-60 µM). Our findings suggest that the antiviral mechanisms primarily involve disruption of host cell protein synthesis and is demonstrably stereoisomer specific. The prospect of a therapeutic window in which emetine or DHE4 inhibit viral propagation without cardiotoxicity renders these alkaloids viable candidates in strategies worthy of clinical investigation.

Screening DNA Damage in the Rat Kidney and Liver by Untargeted DNA Adductomics.

Air pollution, tobacco smoke, and red meat are associated with renal cell cancer (RCC) risk in the United States and Western Europe; however, the chemicals that form DNA adducts and initiate RCC are mainly unknown. Aristolochia herbaceous plants are used for medicinal purposes in Asia and worldwide. They are a significant risk factor for upper tract urothelial carcinoma (UTUC) and RCC to a lesser extent. The aristolochic acid (AA) 8-methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I), a component of Aristolochia herbs, contributes to UTUC in Asian cohorts and in Croatia, where AA-I exposure occurs from ingesting contaminated wheat flour. The DNA adduct of AA-I, 7-(2'-deoxyadenosin-N6-yl)-aristolactam I, is often detected in patients with UTUC, and its characteristic A:T-to-T:A mutational signature occurs in oncogenes and tumor suppressor genes in AA-associated UTUC. Identifying DNA adducts in the renal parenchyma and pelvis caused by other chemicals is crucial to gaining insights into unknown RCC and UTUC etiologies. We employed untargeted screening with wide-selected ion monitoring tandem mass spectrometry (wide-SIM/MS2) with nanoflow liquid chromatography/Orbitrap mass spectrometry to detect DNA adducts formed in rat kidneys and liver from a mixture of 13 environmental, tobacco, and dietary carcinogens that may contribute to RCC. Twenty DNA adducts were detected. DNA adducts of 3-nitrobenzanthrone (3-NBA), an atmospheric pollutant, and AA-I were the most abundant. The nitrophenanthrene moieties of 3-NBA and AA-I undergo reduction to their N-hydroxy intermediates to form 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts. We also discovered a 2'-deoxycytidine AA-I adduct and dA and dG adducts of 10-methoxy-6-nitro-phenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-III), an AA-I isomer and minor component of the herbal extract assayed, signifying AA-III is a potent kidney DNA-damaging agent. The roles of AA-III, other nitrophenanthrenes, and nitroarenes in renal DNA damage and human RCC warrant further study. Wide-SIM/MS2 is a powerful scanning technology in DNA adduct discovery and cancer etiology characterization.

Loss of proximal tubular transcription factor Krüppel-like factor 15 exacerbates kidney injury through loss of fatty acid oxidation.

Loss of fatty acid ß-oxidation (FAO) in the proximal tubule is a critical mediator of acute kidney injury and eventual fibrosis. However, transcriptional mediators of FAO in proximal tubule injury remain understudied. Krüppel-like factor 15 (KLF15), a highly enriched zinc-finger transcription factor in the proximal tubule, was significantly reduced in proximal tubule cells after aristolochic acid I (AAI) treatment, a proximal tubule-specific injury model. Proximal tubule specific knockout of Klf15 exacerbated proximal tubule injury and kidney function decline compared to control mice during the active phase of AAI treatment, and after ischemia-reperfusion injury. Furthermore, along with worsening proximal tubule injury and kidney function decline, knockout mice exhibited increased kidney fibrosis as compared to control mice during the remodeling phase after AAI treatment. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in immune system and integrin signaling pathways and decreased transcripts encompassing metabolic pathways, specifically FAO, and PPARα signaling, in knockout versus control mice after AAI treatment. In silico and experimental chromatin immunoprecipitation studies collectively demonstrated that KLF15 occupied the promoter region of key FAO genes, CPT1A and ACAA2, in close proximity to transcription factor PPARα binding sites. While the loss of Klf15 reduced the expression of Cpt1a and Acaa2 and led to compromised FAO, induction of KLF15 partially rescued loss of FAO in AAI-treated cells. Klf15, Ppara, Cpt1a, and Acaa2 expression was also decreased in other mouse kidney injury models. Tubulointerstitial KLF15 independently correlated with eGFR, PPARA and CPT1A appearance in expression arrays from human kidney biopsies. Thus, proximal tubule-specific loss of Klf15 exacerbates acute kidney injury and fibrosis, likely due to loss of interaction with PPARα leading to loss of FAO gene transcription.

Supervised mutational signatures for obesity and other tissue-specific etiological factors in cancer.

Determining the etiologic basis of the mutations that are responsible for cancer is one of the fundamental challenges in modern cancer research. Different mutational processes induce different types of DNA mutations, providing 'mutational signatures' that have led to key insights into cancer etiology. The most widely used signatures for assessing genomic data are based on unsupervised patterns that are then retrospectively correlated with certain features of cancer. We show here that supervised machine-learning techniques can identify signatures, called SuperSigs, that are more predictive than those currently available. Surprisingly, we found that aging yields different SuperSigs in different tissues, and the same is true for environmental exposures. We were able to discover SuperSigs associated with obesity, the most important lifestyle factor contributing to cancer in Western populations.

Targeted and Untargeted Detection of DNA Adducts of Aromatic Amine Carcinogens in Human Bladder by Ultra-Performance Liquid Chromatography-High-Resolution Mass Spectrometry.

Epidemiological studies have linked aromatic amines (AAs) from tobacco smoke and some occupational exposures with bladder cancer risk. Several epidemiological studies have also reported a plausible role for structurally related heterocyclic aromatic amines present in tobacco smoke or formed in cooked meats with bladder cancer risk. DNA adduct formation is an initial biochemical event in bladder carcinogenesis. We examined paired fresh-frozen (FR) and formalin-fixed paraffin-embedded (FFPE) nontumor bladder tissues from 41 bladder cancer patients for DNA adducts of 4-aminobiphenyl (4-ABP), a bladder carcinogen present in tobacco smoke, and 2-amino-9 H-pyrido[2,3- b]indole, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine and 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline, possible human carcinogens, which occur in tobacco smoke and cooked meats. These chemicals are present in urine of tobacco smokers or omnivores. Targeted DNA adduct measurements were done by ultra-performance liquid chromatography-electrospray ionization multistage hybrid Orbitrap MS. N-(2'-Deoxyguanosin-8-yl)-4-ABP ( N-(dG-C8)-4-ABP) was the sole adduct detected in FR and FFPE bladder tissues. Twelve subjects (29%) had N-(dG-C8)-4-ABP levels above the limit of quantification, ranging from 1.4 to 33.8 adducts per 109 nucleotides (nt). DNA adducts of other human AA bladder carcinogens, including 2-naphthylamine (2-NA), 2-methylaniline (2-MA), 2,6-dimethylaniline (2,6-DMA), and lipid peroxidation (LPO) adducts, were screened for in bladder tissue, by our untargeted data-independent adductomics method, termed wide-selected ion monitoring (wide-SIM)/MS2. Wide-SIM/MS2 successfully detected N-(dG-C8)-4-ABP, N-(2'-deoxyadenosin-8-yl)-4-ABP and the presumed hydrazo linked adduct, N-(2'-deoxyguanosin- N2-yl)-4-ABP, and several LPO adducts in bladder DNA. Wide-SIM/MS2 detected multiple DNA adducts of 2-NA, 2-MA, and, 2,6-DMA, when calf thymus DNA was modified with reactive intermediates of these carcinogens. However, these AA-adducts were below the limit of detection in unspiked human bladder DNA (<1 adduct per 108 nt). Wide-SIM/MS2 can screen for many types of DNA adducts formed with exogenous and endogenous electrophiles and will be employed to identify DNA adducts of other chemicals that may contribute to the etiology of bladder cancer.

Method for Biomonitoring DNA Adducts in Exfoliated Urinary Cells by Mass Spectrometry.

Tobacco smoking contributes to about 50% of the bladder-cancer (BC) cases in the United States. Some aromatic amines in tobacco smoke are bladder carcinogens; however, other causal agents of BC are uncertain. Exfoliated urinary cells (EUCs) are a promising noninvasive biospecimen to screen for DNA adducts of chemicals that damage the bladder genome, although the analysis of DNA adducts in EUCs is technically challenging because of the low number of EUCs and limiting quantity of cellular DNA. Moreover, EUCs and their DNA adducts must remain viable during the time of collection and storage of urine to develop robust screening methods. We employed RT4 cells, a well-differentiated transitional epithelial bladder cell line, as a cell-model system in urine to investigate cell viability and the chemical stability of DNA adducts of two prototypical bladder carcinogens: 4-aminobiphenyl (4-ABP), an aromatic amine found in tobacco smoke, and aristolochic acid I (AA-I), a nitrophenanthrene found in Aristolochia herbaceous plants used for medicinal purposes worldwide. The cell viability of RT4 cells pretreated with 4-ABP or AA-I in urine exceeded 80%, and the major DNA adducts of 4-ABP and AA-I, quantified by liquid chromatography-mass spectrometry, were stable for 24 h. Thereafter, we successfully screened EUCs of mice treated with AA-I to measure DNA adducts of AA-I, which were still detected 25 days following treatment with the carcinogen. EUCs are promising biospecimens that can be employed for the screening of DNA adducts of environmental and dietary genotoxicants that may contribute to the development of BC.

Non-invasive detection of urothelial cancer through the analysis of driver gene mutations and aneuploidy.

Current non-invasive approaches for detection of urothelial cancers are suboptimal. We developed a test to detect urothelial neoplasms using DNA recovered from cells shed into urine. UroSEEK incorporates massive parallel sequencing assays for mutations in 11 genes and copy number changes on 39 chromosome arms. In 570 patients at risk for bladder cancer (BC), UroSEEK was positive in 83% of those who developed BC. Combined with cytology, UroSEEK detected 95% of patients who developed BC. Of 56 patients with upper tract urothelial cancer, 75% tested positive by UroSEEK, including 79% of those with non-invasive tumors. UroSEEK detected genetic abnormalities in 68% of urines obtained from BC patients under surveillance who demonstrated clinical evidence of recurrence. The advantages of UroSEEK over cytology were evident in low-grade BCs; UroSEEK detected 67% of cases whereas cytology detected none. These results establish the foundation for a new non-invasive approach for detection of urothelial cancer.

Human liver-kidney model elucidates the mechanisms of aristolochic acid nephrotoxicity.

Environmental exposures pose a significant threat to human health. However, it is often difficult to study toxicological mechanisms in human subjects due to ethical concerns. Plant-derived aristolochic acids are among the most potent nephrotoxins and carcinogens discovered to date, yet the mechanism of bioactivation in humans remains poorly understood. Microphysiological systems (organs-on-chips) provide an approach to examining the complex, species-specific toxicological effects of pharmaceutical and environmental chemicals using human cells. We microfluidically linked a kidney-on-a-chip with a liver-on-a-chip to determine the mechanisms of bioactivation and transport of aristolochic acid I (AA-I), an established nephrotoxin and human carcinogen. We demonstrate that human hepatocyte-specific metabolism of AA-I substantially increases its cytotoxicity toward human kidney proximal tubular epithelial cells, including formation of aristolactam adducts and release of kidney injury biomarkers. Hepatic biotransformation of AA-I to a nephrotoxic metabolite involves nitroreduction, followed by sulfate conjugation. Here, we identify, in a human tissue-based system, that the sulfate conjugate of the hepatic NQO1-generated aristolactam product of AA-I (AL-I-NOSO3) is the nephrotoxic form of AA-I. This conjugate can be transported out of liver via MRP membrane transporters and then actively transported into kidney tissue via one or more organic anionic membrane transporters. This integrated microphysiological system provides an ex vivo approach for investigating organ-organ interactions, whereby the metabolism of a drug or other xenobiotic by one tissue may influence its toxicity toward another, and represents an experimental approach for studying chemical toxicity related to environmental and other toxic exposures.

A Rapid Throughput Method To Extract DNA from Formalin-Fixed Paraffin-Embedded Tissues for Biomonitoring Carcinogenic DNA Adducts.

Formalin-fixed paraffin-embedded (FFPE) tissues are rarely used for screening DNA adducts of carcinogens because the harsh conditions required to reverse the formaldehyde-mediated DNA cross-links can destroy DNA adducts. We recently adapted a commercial silica-based column kit used in genomics to manually isolate DNA under mild conditions from FFPE tissues of rodents and humans and successfully measured DNA adducts of several carcinogens including aristolochic acid I (AA-I), 4-aminobiphenyl (4-ABP), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (Yun et al. (2013) Anal. Chem. 85, 4251-8, and Guo et al. (2016) Anal. Chem. 88, 4780-7). The DNA retrieval methodology is robust; however, the procedure is time-consuming and labor intensive, and not amenable to rapid throughput processing. In this study, we have employed the Promega Maxwell 16 MDx system, which is commonly used in large scale genomics studies, for the rapid throughput extraction of DNA. This system streamlines the DNA isolation procedure and increases the sample processing rate by about 8-fold over the manual method (32 samples versus 4 samples processed per hour). High purity DNA is obtained in satisfactory yield for the measurements of DNA adducts by ultra performance liquid chromatography-electrospray-ionization-ion trap-multistage scan mass spectrometry. The measurements show that the levels of DNA adducts of AA-I, 4-ABP, and PhIP in FFPE rodent and human tissues are comparable to those levels measured in DNA from matching tissues isolated by the commercial silica-based column kits and in DNA from fresh frozen tissues isolated by the conventional phenol-chloroform extraction method. The isolation of DNA from tissues is one major bottleneck in the analysis of DNA adducts. This rapid throughput methodology greatly decreases the time required to process DNA and can be employed in large-scale epidemiology studies designed to assess the role of chemical exposures and DNA adducts in cancer risk.

Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing.

We present the bottleneck sequencing system (BotSeqS), a next-generation sequencing method that simultaneously quantifies rare somatic point mutations across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately before library amplification. We use BotSeqS to show age- and tissue-dependent accumulations of rare mutations and demonstrate that somatic mutational burden in normal human tissues can vary by several orders of magnitude, depending on biologic and environmental factors. We further show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, the mutation spectra of normal tissues were different from each other, but similar to those of the cancers that arose in them. This technology can provide insights into the number and nature of genetic alterations in normal tissues and can be used to address a variety of fundamental questions about the genomes of diseased tissues.

Aristolochic Acid in the Etiology of Renal Cell Carcinoma.

BACKGROUND: Aristolochia species used in the practice of traditional herbal medicine contains aristolochic acid (AA), an established human carcinogen contributing to urothelial carcinomas of the upper urinary tract. AA binds covalently to genomic DNA, forming aristolactam (AL)-DNA adducts. Here we investigated whether AA is also an etiologic factor in clear cell renal cell carcinoma (ccRCC). METHODS: We conducted a population-based case-control study to investigate the linkage between Aristolochia prescription history, cumulative AA consumption, and ccRCC incidence in Taiwan (5,709 cases and 22,836 matched controls). The presence and level of mutagenic dA-AL-I adducts were determined in the kidney DNA of 51 Taiwanese ccRCC patients. The whole-exome sequences of ccRCC tumors from 10 Taiwanese ccRCC patients with prior exposure to AA were determined. RESULTS: Cumulative ingestion of more than 250 mg of AA increased risk of ccRCC (OR, 1.25), and we detected dA-AL-I adducts in 76% of Taiwanese ccRCC patients. Furthermore, the distinctive AA mutational signature was evident in six of 10 sequenced ccRCC exomes from Taiwanese patients. CONCLUSIONS: This study strongly suggests that AA contributes to the etiology of certain RCCs. IMPACT: The current study offers compelling evidence implicating AA in a significant fraction of the RCC arising in Taiwan and illustrates the power of integrating epidemiologic, molecular, and genetic data in the investigation of cancer etiology. Cancer Epidemiol Biomarkers Prev; 25(12); 1600-8. ©2016 AACR.

Mutational signature of aristolochic acid: Clue to the recognition of a global disease.

Mutational signatures associated with specific forms of DNA damage have been identified in several forms of human cancer. Such signatures provide information regarding mechanisms of tumor induction which, in turn, can reduce exposure to carcinogens by shaping public health policy. Using a molecular epidemiologic approach that takes advantage of recent advances in genome sequencing while applying sensitive and specific analytical methods to characterize DNA damage, it has become increasingly possible to establish causative linkages between certain environmental mutagens and disease risk. In this perspective, we use aristolochic acid, a human carcinogen and nephrotoxin found in Aristolochia herbs, to illustrate the power and effectiveness of this multidisciplinary approach. The genome-wide mutational signature for this toxin, detected initially in cancers of the upper urinary tract, has subsequently been associated with cancers of the liver and kidney. These findings have significant implications for global public health, especially in China, where millions of individuals have used Aristolochia herbal remedies as part of traditional Chinese medicine and, thus, are at risk of developing aristolochic acid nephropathy and/or upper urinary tract carcinomas. The studies reported here set the stage for research into prevention and early detection, both of which will be required to manage a potentially devastating global disease.

Multiclass Carcinogenic DNA Adduct Quantification in Formalin-Fixed Paraffin-Embedded Tissues by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

DNA adducts are a measure of internal exposure to genotoxicants and an important biomarker for human risk assessment. However, the employment of DNA adducts as biomarkers in human studies is often restricted because fresh-frozen tissues are not available. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis are readily accessible. Recently, our laboratory reported that DNA adducts of aristolochic acid, a carcinogenic component of Aristolochia herbs used in traditional Chinese medicines worldwide, can be recovered quantitatively from FFPE tissues. In this study, we have evaluated the efficacy of our method for retrieval of DNA adducts from archived tissue by measuring DNA adducts derived from four other classes of human carcinogens: polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic aromatic amines (HAAs), and N-nitroso compounds (NOCs). Deoxyguanosine (dG) adducts of the PAH benzo[a]pyrene (B[a]P), 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N(2)-B[a]PDE); the aromatic amine 4-aminobiphenyl (4-ABP), N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP); the HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP); and the dG adducts of the NOC 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), O(6)-methyl-dG (O(6)-Me-dG) and O(6)-pyridyloxobutyl-dG (O(6)-POB-dG), formed in liver, lung, bladder, pancreas, or colon were recovered in comparable yields from fresh-frozen and FFPE preserved tissues of rodents treated with the procarcinogens. Quantification was achieved by ultraperformance liquid chromatography coupled with electrospray ionization ion-trap multistage mass spectrometry (UPLC/ESI-IT-MS(3)). These advancements in the technology of DNA adduct retrieval from FFPE tissue clear the way for use of archived pathology samples in molecular epidemiology studies designed to assess the causal role of exposure to hazardous chemicals with cancer risk.

New Approaches for Biomonitoring Exposure to the Human Carcinogen Aristolochic Acid.

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.

Formalin-fixed paraffin-embedded tissue as a source for quantitation of carcinogen DNA adducts: aristolochic acid as a prototype carcinogen.

DNA adducts are a measure of internal exposure to genotoxicants. However, the measurement of DNA adducts in molecular epidemiology studies often is precluded by the lack of fresh tissue. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues frequently are accessible, although technical challenges remain in retrieval of high quality DNA suitable for biomonitoring of adducts. Aristolochic acids (AA) are human carcinogens found in Aristolochia plants, some of which have been used in the preparation of traditional Chinese herbal medicines. We previously established a method to measure DNA adducts of AA in FFPE tissue. In this study, we examine additional features of formalin fixation that could impact the quantity and quality of DNA and report on the recovery of AA-DNA adducts in mice exposed to AA. The yield of DNA isolated from tissues fixed with formalin decreased over 1 week; however, the levels of AA-DNA adducts were similar to those in fresh frozen tissue. Moreover, DNA from FFPE tissue served as a template for PCR amplification, yielding sequence data of comparable quality to DNA obtained from fresh frozen tissue. The estimates of AA-DNA adducts measured in freshly frozen tissue and matching FFPE tissue blocks of human kidney stored for 9 years showed good concordance. Thus, DNA isolated from FFPE tissues may be used to biomonitor DNA adducts and to amplify genes used for mutational analysis, providing clues regarding the origin of human cancers for which an environmental cause is suspected.

Mutational signature of aristolochic acid exposure as revealed by whole-exome sequencing.

In humans, exposure to aristolochic acid (AA) is associated with urothelial carcinoma of the upper urinary tract (UTUC). Exome sequencing of UTUCs from 19 individuals with documented exposure to AA revealed a remarkably large number of somatic mutations and an unusual mutational signature attributable to AA. Most of the mutations (72%) in these tumors were A:T-to-T:A transversions, located predominantly on the nontranscribed strand, with a strong preference for deoxyadenosine in a consensus sequence (T/CAG). This trinucleotide motif overlaps the canonical splice acceptor site, possibly accounting for the excess of splice site mutations observed in these tumors. The AA mutational fingerprint was found frequently in oncogenes and tumor suppressor genes in AA-associated UTUC. The AA mutational signature was observed in one patient's tumor from a UTUC cohort without previous indication of AA exposure. Together, these results directly link an established environmental mutagen to cancer through genome-wide sequencing and highlight its power to reveal individual exposure to carcinogens.

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 Aristolochia 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-d]-1,3-dioxole-5-carboxylic acid (AA-I) is a component of Aristolochia herbs and a potent human urothelial carcinogen. AA-I reacts with DNA to form the aristolactam (AL-I)-DNA adduct 7-(deoxyadenosin-N(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 10(9) 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.

TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal.

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.