Abstract Aristolochic acids (AA) are human carcinogens and nephrotoxins found in Aristolochia plants, including herbs used in traditional medicines throughout the world. Aristolochic acids can also be ingested as an environmental contaminant in food, which forms the basis of Balkan endemic nephropathy and associated upper tract urothelial cancer. Aristolactam DNA-adducts, formed after metabolism of AA, are promutagenic and are resistant to repair and readily detected in human tissue sample DNA decades after exposure. Thus, AL-DNA adducts are excellent biomarkers of exposure to this carcinogen. The mutational signature of AA is distinct for an environmental carcinogen and is recognized as Signature 22 in the COSMIC mutational signature database. Molecular epidemiology using the presence of adducts and/or the presence of Signature 22 has implicated AA in the etiology of several cancers, including upper-tract urothelial cancer, bladder cancer, renal cell clear cell carcinoma, hepatocellular carcinoma, and cholangiocarcinomas. In regions of the world reliant on traditional medicines, including much of Asia, the AA contribution to incidence of these cancers is significant. In the Balkan countries of Europe, the route of exposure is thought to occur via contamination of wheat harvests with Aristolochia plant material from weed growth. In endemic villages farming families traditionally have ingested primarily wheat flour milled from their own harvests. An alternative hypothesis is that decaying Aristolochia plants release the chemically stable aristolochic acids into the soil, and these are then taken up and concentrated by food plants. To distinguish between these hypotheses, we have obtained 25 flour samples from individual farms along the Croatia/Bosnia and Herzegovina border. To identify AA-contaminated flour, aristolochic acids were extracted from aliquots of the flour, reduced to aristolactams, and quantitated with an LC-ESI-MS/MS method. Several flour samples were indeed contaminated with AA in the range of 10–100 parts per billion. Secondly, we also extracted DNA from each flour sample. The direct contamination hypothesis predicts the presence of Aristolochia DNA in contaminated flour samples; the indirect contamination via decay hypothesis does not. Using widely accepted plant “barcode” PCR protocols, we amplified a portion of the rbcLa gene with generic primers that amplify the gene from all species. Within this PCR amplicon each plant species has a constellation of specific base-pairs. Thus, the contribution of all plant species to the flour sample can be determined by highly parallel sequencing of the amplicon. We will present the results of this next-gen sequencing to determine the contribution of Aristolochia to Balkan flour samples and correlation with aristolochic acid content. Citation Format: Viktoriya Sidorenko, Robert Rieger, Bojan Jelakovic, Thomas Rosenquist. Dietary ingestion of aristolochic acid: Mechanisms of exposure [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A20.

Lipid homeostasis allows cells to adjust membrane biophysical properties in response to changes in environmental conditions. In the yeast Saccharomyces cerevisiae, a downward shift in temperature from an optimal reduces membrane fluidity, which triggers a lipid remodeling of the plasma membrane. How changes in membrane fluidity are perceived, and how the abundance and composition of different lipid classes is properly balanced, remain largely unknown. Here, we show that the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], the most abundant plasma membrane phosphoinositide, drop rapidly in response to a downward shift in temperature. This change triggers a signaling cascade transmitted to cytosolic diphosphoinositol phosphate derivatives, among them 5-PP-IP4 and 1-IP7, that exert regulatory functions on genes involved in the inositol and phospholipids (PLs) metabolism, and inhibit the activity of the protein kinase Pho85. Consistent with this, cold exposure triggers a specific program of neutral lipids and PLs changes. Furthermore, we identified Pho85 as playing a key role in controlling the synthesis of long-chain bases (LCBs) via the Ypk1-Orm2 regulatory circuit. We conclude that Pho85 orchestrates a coordinated response of lipid metabolic pathways that ensure yeast thermal adaptation.

Aristolochic acid (AA) is a potent human carcinogen and nephrotoxin found in preparations of Aristolochia plants used in Chinese Traditional Medicine. Following biotransformation to form N-sulfonyloxyaristolactam (AL-I-NOSO3), this intermediate undergoes heterolytic cleavage of the sulfate group to generate a reactive cyclic nitrenium ion, the ultimate DNA binding species. Recently, we showed that primary human hepatocytes significantly increase renal toxicity of AA in the integrated human liver-kidney “organs on-chips” model. Therefore, we propose that AA is activated in the liver by forming AL-I-NOSO3, which is transported to the kidney protected from decomposition by binding to serum albumin. We employed mass spectrometric, fluorimetric and X-crystallography based approaches to dissect mechanisms of interactions between human serum albumin (HSA), AA, N-hydroxyaristolactam and AL-I-NOSO3. First, we demonstrate that HSA stabilizes otherwise labile N-sulfonyloxyaristolactam. Quenching of the native fluorescence of HSA due to the presence of a sole molecule of tryptophane-214, allowed us to conclude that all three compounds have similar affinities to IIA drug binding pocket of HSA. Subsequently, we obtained a high-resolution X-ray structure of AA bound to HSA in domain IB (1.9°A, pdb: 6HSC). Since prior to crystallization HSA was enriched with sodium myristate and site IB in circulation is occupied by fatty acids, our results imply that the IB pocket is the primary high affinity binding site for AA and its active forms. To assess whether AL-I-NOSO3 covalently binds protein, we incubated human plasma and purified HSA with this active AA. Immunoblotting of reacted HSA using antibodies that recognize aristolactam(AL)-adducted DNA suggests irreversible covalent adduction of AL to HSA. A combined approach using mass spectrometry instruments and enzymatic digestion revealed that AL is adducted to HSA at the following sites: Trp-214, Tyr-138 and Tyr-141. The former amino acid is located in the IIA drug binding site of HSA, while the latter two can be found in our HSA/AA structure in the site IB in the vicinity to AA molecule, corroborating our X-crystallography and fluoremetric data. Based on these studies we propose that AL-I-NOSO3 has a dual mode of interactions with HSA. If AL-I-NOSO3 decomposes prior to HSA binding, aristolactam will become irreversibly trapped with HSA. This binding to HSA would serve as mechanism of detoxication of AA species. However, if AL-I-NOSO3 binds to HSA prior to decomposition, it should be protected by HSA and transported to target tissues in its intact form. Citation Format: Sergei Pomyalov, Radha Bonala, Robert Rieger, Irina Zaitseva, Charles Iden, John Haley, Robert Turesky, Francis Johnson, Thomas Rosenquist, Arthur P. Grollman, Gil Shoham, Viktoriya S. Sidorenko. Molecular mechanisms by which a bioactivated human carcinogen is transported to target tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4660.

Cryptococcus neoformans (C. neoformans) is estimated to cause about 220,000 new cases every year in patients with AIDS, despite advances in antifungal treatments. C. neoformans possesses a remarkable ability to disseminate through an immunocompromised host, making treatment difficult. Here, we examine the mechanism of survival of C. neoformans under varying host conditions and find a role for ceramide synthase in C. neoformans virulence. This study also provides a detailed lipidomics resource for the fungal lipid research community in addition to discovering a potential target for antifungal therapy.

The incidence of invasive fungal infections has risen dramatically in recent decades. Current antifungal drugs are either toxic, likely to interact with other drugs, have a narrow spectrum of activity, or induce fungal resistance. Hence, there is a great need for new antifungals, possibly with novel mechanisms of action. Previously our group reported an acylhydrazone called BHBM that targeted the sphingolipid pathway and showed strong antifungal activity against several fungi. In this study, we screened 19 derivatives of BHBM. Three out of 19 derivatives were highly active against Cryptococcus neoformans in vitro and had low toxicity in mammalian cells. In particular, one of them, called D13, had a high selectivity index and showed better activity in an animal model of cryptococcosis, candidiasis, and pulmonary aspergillosis. D13 also displayed suitable pharmacokinetic properties and was able to pass through the blood-brain barrier. These results suggest that acylhydrazones are promising molecules for the research and development of new antifungal agents.

2The Jackson Laboratory, Bar Harbor, ME 04609, USA 3Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh 226007, India 4Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA 5Proteomics Center, Stony Brook University, Stony Brook, NY 11794, USA 6Department of Biomedical Sciences, School of Health Professions and Nursing, Long Island University, Brookville, NY 11548, USA

Gomolin, Irving H. MDCM; Papamichael, Michael J. MD; Fazzari, Melissa J. PhD; Rieger, Robert BS Author Information

Aristolochic acids are natural nitro-compounds found globally in the plant genus Aristolochia 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 O-sulfate, aristolactam Ia, aristolactam I, and aristolactam Ia O-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.

Pyridoxal-5′-phosphate (PLP), in addition to its known metabolic functions, inactivates many DNA-dependent enzymes through conjugation to their critical amino groups. We have investigated the ability of PLP to inhibit bifunctional DNA repair glycosylases, which possess a catalytic amine. Of six enzymes tested, only endonuclease VIII-like protein 2 (NEIL2) was significantly inhibited by PLP. The inhibition was due to Schiff base formation between PLP and the enzyme. PLP-conjugated NEIL2 completely lost its ability to bind damaged DNA. Liquid chromatography/nanoelectrospray ionization tandem mass spectrometry of the products of proteolysis of pyridoxylated NEIL2 identified Lys50 as the site of modification. Thus, the β2/β3 loop where Lys50 is located in NEIL2 is important for DNA binding, presumably lies next to a phosphate-binding site, and may represent a target for regulation of the enzyme activity.

Ingestion of aristolochic acids (AA) contained in herbal remedies results in aristolochic acid nephropathy (AAN), which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA I and AA II, primary components in AA, have similar genotoxic potential, whereas only AA I shows severe renal toxicity in rodents. AA I is demethylated to form 8‐hydroxy‐aristolochic acid I (AA Ia) as a major metabolite. However, the nephrotoxicity and genotoxicity of AA Ia has not yet been determined. AA Ia was isolated from urine collected from rats treated with AA I and characterized by NMR and mass spectrometry. The purified AA Ia was administered intraperitoneally to C3H/He male mice for 9 days and its toxicity was compared with AA I. Using 32 P‐postlabeling/polyacrylamide gel electrophoresis, the level of AA Ia‐derived DNA adducts in renal cortex was ∼70–110 times lower than that observed with AA I, indicating that AA Ia has only a limited genotoxicity. Supporting this result, when calf thymus DNA was reacted with AA Ia in a buffer containing zinc dust, the formation of AA Ia‐DNA adducts was two‐orders of magnitude lower than that of AA I. Histopathologic analysis revealed that unlike AA I, no significant changes were detected in the renal cortex of mice treated with AA Ia. Therefore, the contribution of AA Ia to renal toxicity is minimum. We conclude the metabolic pathway of converting AA I to AA Ia functions as the detoxification of AA I.

Future treatments for chronic hepatitis C virus (HCV) infection are likely to include agents that target viral components directly. Here, the preclinical characteristics of ITMN-191, a peptidomimetic inhibitor of the NS3/4A protease of HCV, are described. ITMN-191 inhibited a reference genotype 1 NS3/4A protein in a time-dependent fashion, a hallmark of an inhibitor with a two-step binding mechanism and a low dissociation rate. Under preequilibrium conditions, 290 pM ITMN-191 half-maximally inhibited the reference NS3/4A protease, but a 35,000-fold-higher concentration did not appreciably inhibit a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Subnanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 4, 5, and 6, while single-digit nanomolar potency was observed against NS3/4A from genotypes 2b and 3a. Dilution of a preformed enzyme inhibitor complex indicated ITMN-191 remained bound to and inhibited NS3/4A for more than 5 h after its initial association. In cell-based potency assays, half-maximal reduction of genotype 1b HCV replicon RNA was afforded by 1.8 nM; 45 nM eliminated the HCV replicon from cells. Peginterferon alfa-2a displayed a significant degree of antiviral synergy with ITMN-191 and reduced the concentration of ITMN-191 required for HCV replicon elimination. A 30-mg/kg of body weight oral dose administered to rats or monkeys yielded liver concentrations 12 h after dosing that exceeded the ITMN-191 concentration required to eliminate replicon RNA from cells. These preclinical characteristics compare favorably to those of other inhibitors of NS3/4A in clinical development and therefore support the clinical investigation of ITMN-191 for the treatment of chronic hepatitis C.

Endemic (Balkan) nephropathy (EN), a devastating renal disease affecting men and women living in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia, is characterized by its insidious onset, invariable progression to chronic renal failure and a strong association with transitional cell (urothelial) carcinoma of the upper urinary tract. Significant epidemiologic features of EN include its focal occurrence in certain villages and a familial, but not inherited, pattern of disease. Our experiments test the hypothesis that chronic dietary poisoning by aristolochic acid is responsible for EN and its associated urothelial cancer. Using 32P-postlabeling/PAGE and authentic standards, we identified dA-aristolactam (AL) and dG-AL DNA adducts in the renal cortex of patients with EN but not in patients with other chronic renal diseases. In addition, urothelial cancer tissue was obtained from residents of endemic villages with upper urinary tract malignancies. The AmpliChip p53 microarray was then used to sequence exons 2–11 of the p53 gene where we identified 19 base substitutions. Mutations at A:T pairs accounted for 89% of all p53 mutations, with 78% of these being A:T → T:A transversions. Our experimental results, namely, that (i) DNA adducts derived from aristolochic acid (AA) are present in renal tissues of patients with documented EN, (ii) these adducts can be detected in transitional cell cancers, and (iii) A:T → T:A transversions dominate the p53 mutational spectrum in the upper urinary tract malignancies found in this population lead to the conclusion that dietary exposure to AA is a significant risk factor for EN and its attendant transitional cell cancer.

Background — It has been established that hyperhomocyst(e)inemia (HHCy) is an independent and graded risk factor for atherosclerosis, although the molecular link to the atherosclerotic process remains obscure. Methods and Results — Screening human umbilical vein endothelial cells (HUVECs) with complementary DNA microarray for the gene expression modified by homocysteine (Hcy) revealed that 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) was upregulated. This effect was confirmed using quantitative reverse transcriptase-polymerase chain reaction. Actinomycin D studies revealed that Hcy stabilized HMGCR mRNA (τ 1/2 , 9.5±1.0 versus 5.0±0.2 hours). Expression of immunodetectable HMGCR in both HUVECs and renal microvascular endothelial cells was increased in Hcy-treated cells in association with the increased abundance of caveolin. Application of a cell-permeable superoxide dismutase mimetic, Mn-TBAP, reversed the Hcy-induced expression of HMGCR. Additional biochemical analysis of the abundance of total cellular cholesterol showed that 0, 20, 50, and 100 μmol/L Hcy resulted in 22.2±7.3%, 39.5±1.2%, and 50.4±6.8% increase, respectively. Gas chromatography mass spectrometry analysis of extracted cholesterol from Hcy-treated HUVECs and from the culture medium showed 17.8±5.2% and 24.0±14.5% increases, respectively. Application of simvastatin to Hcy-treated cells reduced cellular cholesterol and prevented Hcy-induced suppression of NO production by HUVECs in a dose-dependent manner. Conclusions — Using a cDNA microarray, the data disclosed an unexpected link between Hcy and cholesterol dysregulation based on the finding of increased abundance of HMGCR mRNA and protein in endothelial cells, demonstrated the possible role of Hcy-induced oxidative stress in this response, and revealed the improvement of endothelial NO production in Hcy-treated HUVECs by statins. Collectively, these findings may provide a solid explanation for the observed proatherogenic effect of HHcy.

Treatment with tamoxifen increased the risk of endometrial cancers in breast cancer patients and women participating in the chemoprevention study. In our laboratory, tamoxifen-DNA adducts, including alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG-N(2)-TAM), were detected in the endometrium of women taking tamoxifen [Shibutani, S., et al. (1999) Chem. Res. Toxicol. 12, 646-653]. On the basis of recent animal studies, deoxyguanosinyl-N-desmethyltamoxifen (dG-N-desmethylTAM) adducts are also suspected to be formed in the liver. In the study presented here, we synthesized alpha-acetoxy-N-desmethyltamoxifen as a model activated metabolite of N-desmethyltamoxifen. The overall yield of alpha-acetoxy-N-desmethyltamoxifen from alpha-hydroxytamoxifen was approximately 42%. alpha-Acetoxy-N-desmethyltamoxifen was highly reactive to 2'-deoxyguanosine, as was similarly observed for tamoxifen alpha-sulfate. The two reaction products were identified as a mixture of epimers of the trans form or cis form of alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG-N(2)-N-desmethylTAM) by mass and proton magnetic resonance spectroscopy. In addition, the trans and cis forms of dG 3'-monophosphate-N(2)-N-desmethylTAM were prepared as standard markers for (32)P-postlabeling/HPLC analysis. Using this technique, dG-N(2)-N-desmethylTAM adducts were detected in calf thymus DNA reacted with alpha-acetoxy-N-desmethyltamoxifen.

Electrospray mass spectrometry techniques were used to characterize components of the active site in Endonuclease VIII by identifying the amino acid sequence and the binding site for a tryptic peptide derived from Endo VIII in a cross-linked DNA-peptide complex. Endo VIII, a DNA repair enzyme with both glycosylase and lyase activities, was covalently bound to a thymidine glycol-containing oligodeoxynucleotide duplex by converting a transient Schiff base formed during the course of the glycosylase activity to a stable covalent bond by chemical reduction with sodium borohydride. After tryptic digestion of the initial product, the identification of the cross-linked peptide was deduced initially from the molecular mass of the tryptic product obtained by negative ion electrospray mass analysis. Nanospray tandem mass spectrometry (MS/MS) analysis of the tryptic product corroborated the molecular mass of the peptide fragment and verified the point of attachment to the oligomer, but failed to produce sufficient fragmentation to sequence the peptide completely. Direct evidence for the amino acid sequence of the peptide was obtained after enzymatic digestion of the DNA portion of the cross-linked DNA-peptide product and analysis by negative ion nanospray MS/MS. Examination of the ions from collision induced fragmentation disclosed that this substance was the N-terminal tryptic fragment of Endo VIII cross-linked to a portion of the oligomer, and that the N-terminal proline from Endo VIII was covalently bound to the residual deoxyribose moiety at the original location of the thymine glycol in the oligomer.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSite-specific adduct formation in oligomeric DNA using a new protecting groupFrancis Johnson, Ivan Habus, Robert G. Gentles, Shinya Shibutani, Hong Chang Lee, Charles R. Iden, and Robert RiegerCite this: J. Am. Chem. Soc. 1992, 114, 12, 4923–4924Publication Date (Print):June 1, 1992Publication History Published online1 May 2002Published inissue 1 June 1992https://pubs.acs.org/doi/10.1021/ja00038a079https://doi.org/10.1021/ja00038a079research-articleACS PublicationsRequest reuse permissionsArticle Views99Altmetric-Citations12LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts