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Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSA MYR ) and its AA complexes (HSA MYR /AA-I and HSA MYR /AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 10 6  M -1 for HSA/AA-I versus 8.4 and 9.0 · 10 5  M -1 for HSA/AA-II. Fluorescence quenching of the HSA Trp 214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., IC 50 ∼ 50-100 nM), paralleling those required for inhibition of protein synthesis, while calcium channel blocking activity occurs at elevated concentrations (i.e., IC 50 ∼ 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Proximal tubular uptake of aristolochic acid (AA) forms aristolactam (AL)-DNA adducts, which cause a p53/p21-mediated DNA damage response and acute tubular injury. Recurrent AA exposure causes kidney function loss and fibrosis in humans (Balkan endemic nephropathy) and mice and is a model of (acute kidney injury) AKI to chronic kidney disease (CKD) transition. Inhibitors of the proximal tubule sodium-glucose transporter SGLT2 can protect against CKD progression, but their effect on AA-induced kidney injury remains unknown. C57BL/6J mice (15-wk-old) were administered vehicle or AA every 3 days for 3 wk (10 and 3 mg/kg ip in females and males, respectively). Dapagliflozin (dapa, 0.01 g/kg diet) or vehicle was initiated 7 days prior to AA injections. All dapa effects were sex independent, including a robust glycosuria. Dapa lowered urinary kidney-injury molecule 1 (KIM-1) and albumin (both normalized to creatinine) after the last AA injection and kidney mRNA expression of early DNA damage response markers ( p53 and p21 ) 3 wk later at the study end. Dapa also attenuated AA-induced increases in plasma creatinine as well as AA-induced up-regulation of renal pro-senescence, pro-inflammatory and pro-fibrotic genes, and kidney collagen staining. When assessed 1 day after a single AA injection, dapa pretreatment attenuated AL-DNA adduct formation by 10 and 20% in kidney and liver, respectively, associated with reduced p21 expression. Initiating dapa application after the last AA injection also improved kidney outcome but in a less robust manner. In conclusion, the first evidence is presented that pretreatment with an SGLT2 inhibitor can attenuate the AA-induced DNA damage response and subsequent nephropathy. NEW & NOTEWORTHY Recurrent exposure to aristolochic acid (AA) causes kidney function loss and fibrosis in mice and in humans, e.g., in the form of the endemic Balkan nephropathy. Inhibitors of the proximal tubule sodium-glucose transporter SGLT2 can protect against CKD progression, but their effect on AA-induced kidney injury remains unknown. Here we provide the first evidence in a murine model that pretreatment with an SGLT2 inhibitor can attenuate the AA-induced DNA damage response and subsequent nephropathy.

In an effort to identify functional-energetic correlations leading to the development of efficient anti-SARS-CoV-2 therapeutic agents, we have designed synthetic analogs of aurintricarboxylic acid (ATA), a heterogeneous polymeric mixture of structurally related linear homologs known to exhibit a host of biological properties, including antiviral activity. These derivatives are evaluated for their ability to interact with a plasma transporter protein (human serum albumin), eukaryotic (yeast) ribosomes, and a SARS-CoV-2 target, the RNA-dependent RNA polymerase (RdRp). The resultant data are critical for characterizing drug distribution, bioavailability, and effective inhibition of host and viral targets. Promising lead compounds are selected on the basis of their binding energetics which have been characterized and correlated with functional activities as assessed by inhibition of RNA replication and protein synthesis. Our results reveal that the activity of heterogeneous ATA is mimicked by linear compounds of defined molecular weight, with a dichlorohexamer salicylic-acid derivative exhibiting the highest potency. These findings are instrumental for optimizing the design of structurally defined ATA analogs that fulfill the requirements of an antiviral drug with respect to bioavailability, homogeneity, and potency, thereby expanding the arsenal of therapeutic regimens that are currently available to address the urgent need for effective SARS-CoV-2 treatment strategies.

Altered cellular metabolism in kidney proximal tubule (PT) cells plays a critical role in acute kidney injury (AKI). The transcription factor Krüppel-like factor 6 (KLF6) is rapidly and robustly induced early in the PT after AKI. We found that PT-specific Klf6 knockdown ( Klf6 PTKD ) is protective against AKI and kidney fibrosis in mice. Combined RNA and chromatin immunoprecipitation sequencing analysis demonstrated that expression of genes encoding branched-chain amino acid (BCAA) catabolic enzymes was preserved in Klf6 PTKD mice, with KLF6 occupying the promoter region of these genes. Conversely, inducible KLF6 overexpression suppressed expression of BCAA genes and exacerbated kidney injury and fibrosis in mice. In vitro , injured cells overexpressing KLF6 had similar decreases in BCAA catabolic gene expression and were less able to utilize BCAA. Furthermore, knockdown of BCKDHB , which encodes one subunit of the rate-limiting enzyme in BCAA catabolism, resulted in reduced ATP production, while treatment with BCAA catabolism enhancer BT2 increased metabolism. Analysis of kidney function, KLF6 , and BCAA gene expression in human chronic kidney disease patients showed significant inverse correlations between KLF6 and both kidney function and BCAA expression. Thus, targeting KLF6-mediated suppression of BCAA catabolism may serve as a key therapeutic target in AKI and kidney fibrosis., Competing Interests: Competing interest statement: J.C.H. declares a Shangpharma research grant of $250,000 per year in 2019 to 2020 and is a Scientific Advisory Board member for RenalytixAI.

Background: Aristolochic acids (AA) and arsenic are chemical carcinogens associated with urothelial carcinogenesis. Here we investigate the combined effects of AA and arsenic toward the risk of developing upper tract urothelial carcinoma (UTUC)., Methods: Hospital-based ( n = 89) and population-based (2,921 cases and 11,684 controls) Taiwanese UTUC cohorts were used to investigate the association between exposure to AA and/or arsenic and the risk of developing UTUC. In the hospital cohort, AA exposure was evaluated by measuring aristolactam-DNA adducts in the renal cortex and by identifying A>T TP53 mutations in tumors. In the population cohort, AA exposure was determined from prescription health insurance records. Arsenic levels were graded from 0 to 3 based on concentrations in well water and the presence of arseniasis-related diseases., Results: In the hospital cohort, 43, 26, and 20 patients resided in grade 0, 1+2, and 3 arseniasis-endemic areas, respectively. Aristolactam-DNA adducts were present in >90% of these patients, indicating widespread AA exposure. A>T mutations in TP53 were detected in 28%, 44%, and 22% of patients residing in grade 0, 1+2, and 3 arseniasis-endemic areas, respectively. Population studies revealed that individuals who consumed more AA-containing herbs had a higher risk of developing UTUC in both arseniasis-endemic and nonendemic areas. Logistic regression showed an additive effect of AA and arsenic exposure on the risk of developing UTUC., Conclusions: Exposure to both AA and arsenic acts additively to increase the UTUC risk in Taiwan., Impact: This is the first study to investigate the combined effect of AA and arsenic exposure on UTUC., (©2020 American Association for Cancer Research.)

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.

Aristolochic acids (AA) are implicated in the development of chronic renal disease and upper urinary tract carcinoma in humans. Using in vitro approaches, we demonstrated that N-hydroxyaristolactams, metabolites derived from partial nitroreduction of AA, require sulfotransferase (SULT)-catalyzed conjugation with a sulfonyl group to form aristolactam-DNA adducts. Following up on this observation, bioactivation of AA-I and N-hydroxyaristolactam I (AL-I-NOH) was studied in human kidney (HK-2) and skin fibroblast (GM00637) cell lines. Pentachlorophenol, a known SULT inhibitor, significantly reduced cell death and aristolactam-DNA adduct levels in HK-2 cells following exposure to AA-I and AL-I-NOH, suggesting a role for Phase II metabolism in AA activation. A gene knockdown, siRNA approach was employed to establish the involvement of selected SULTs and nitroreductases in AA-I bioactivation. Silencing of SULT1A1 and PAPSS2 led to a significant decrease in aristolactam-DNA levels in both cell lines following exposure to AA-I, indicating the critical role for sulfonation in the activation of AA-I in vivo Since HK-2 cells proved relatively resistant to knockdown with siRNAs, gene silencing of xanthine oxidoreductase, cytochrome P450 oxidoreductase and NADPH:quinone oxidoreductase was conducted in GM00637 cells, showing a significant increase, decrease and no effect on aristolactam-DNA levels, respectively. In GM00637 cells exposed to AL-I-NOH, suppressing the SULT pathway led to a significant decrease in aristolactam-DNA formation, mirroring data obtained for AA-I. We conclude from these studies that SULT1A1 is involved in the bioactivation of AA-I through the sulfonation of AL-I-NOH, contributing significantly to the toxicities of AA observed in vivo., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urologic cancers. Monitoring of AA exposure uses biomarkers such as aristolactam-DNA adducts, detected by mass spectrometry in the kidney cortex, or the somatic A>T transversion pattern characteristic of exposure to AA, as revealed by previous DNA-sequencing studies using fresh-frozen tumors., Methods: Here, we report a low-coverage whole-exome sequencing method (LC-WES) optimized for multisample detection of the AA mutational signature, and demonstrate its utility in 17 formalin-fixed paraffin-embedded urothelial tumors obtained from 15 patients with endemic nephropathy, an environmental form of AA nephropathy., Results: LC-WES identified the AA signature, alongside signatures of age and APOBEC enzyme activity, in 15 samples sequenced at the average per-base coverage of approximately 10×. Analysis at 3 to 9× coverage revealed the signature in 91% of the positive samples. The exome-wide distribution of the predominant A>T transversions exhibited a stochastic pattern, whereas 83 cancer driver genes were enriched for recurrent nonsynonymous A>T mutations. In two patients, pairs of tumors from different parts of the urinary tract, including the bladder, harbored overlapping mutation patterns, suggesting tumor dissemination via cell seeding., Conclusions: LC-WES analysis of archived tumor tissues is a reliable method applicable to investigations of both the exposure to AA and its biologic effects in human carcinomas., Impact: By detecting cancers associated with AA exposure in high-risk populations, LC-WES can support future molecular epidemiology studies and provide evidence-base for relevant preventive measures., (©2015 American Association for Cancer Research.)

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/MS n ). 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/MS n surpasses the limits of detection of AL-DNA adducts obtained by 32 P-postlabeling techniques, the most widely employed methods for detecting putative DNA adducts in humans. AL-DNA adducts can be measured by UPLC-ESI/MS 3 , 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.

Aristolochic acids are potent human carcinogens; the role of phase II metabolism in their bioactivation is unclear. Accordingly, we tested the ability of the partially reduced metabolites, N-hydroxyaristolactams (AL-NOHs), and their N-O-sulfonated and N-O-acetylated derivatives to react with DNA to form aristolactam-DNA adducts. AL-NOHs displayed little or no activity in this regard while the sulfo- and acetyl compounds readily form DNA adducts, as detected by (32)P-post-labeling analysis. Mouse hepatic and renal cytosols stimulated binding of AL-NOHs to DNA in the presence of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) but not of acetyl-CoA. Using Time of Flight liquid chromatography/mass spectrometry, N-hydroxyaristolactam I formed the sulfated compound in the presence of PAPS and certain human sulfotransferases, SULT1B1 >>> SULT1A2 > SULT1A1 >>> SULT1A3. The same pattern of SULT reactivity was observed when N-hydroxyaristolactam I was incubated with these enzymes and PAPS and the reaction was monitored by formation of aristolactam-DNA adducts. In the presence of human, Nad(p)h: quinone oxidoreductase, the ability of aristolochic acid I to bind DNA covalently was increased significantly by addition of PAPS and SULT1B1. We conclude from these studies that AL-NOHs, formed following partial nitroreduction of aristolochic acids, serve as substrates for SULT1B1, producing N-sulfated esters, which, in turn, are converted to highly active species that react with DNA and, potentially, cellular proteins, resulting in the genotoxicity and nephrotoxicity associated with ingestion of aristolochic acids by humans., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aristolochic acid, a potent human carcinogen produced by Aristolochia plants, is associated with urothelial carcinoma of the upper urinary tract (UUC). Following metabolic activation, aristolochic acid reacts with DNA to form aristolactam (AL)-DNA adducts. These lesions concentrate in the renal cortex, where they serve as a sensitive and specific biomarker of exposure, and are found also in the urothelium, where they give rise to a unique mutational signature in the TP53 tumor-suppressor gene. Using AL-DNA adducts and TP53 mutation spectra as biomarkers, we conducted a molecular epidemiologic study of UUC in Taiwan, where the incidence of UUC is the highest reported anywhere in the world and where Aristolochia herbal remedies have been used extensively for many years. Our study involves 151 UUC patients, with 25 patients with renal cell carcinomas serving as a control group. The TP53 mutational signature in patients with UUC, dominated by otherwise rare A:T to T:A transversions, is identical to that observed in UUC associated with Balkan endemic nephropathy, an environmental disease. Prominent TP53 mutational hotspots include the adenine bases of (5')AG (acceptor) splice sites located almost exclusively on the nontranscribed strand. A:T to T:A mutations also were detected at activating positions in the FGFR3 and HRAS oncogenes. AL-DNA adducts were present in the renal cortex of 83% of patients with A:T to T:A mutations in TP53, FGFR3, or HRAS. We conclude that exposure to aristolochic acid contributes significantly to the incidence of UUC in Taiwan, a finding with significant implications for global public health.

Exposure to aristolochic acid (AA), a component of Aristolochia plants used in herbal remedies, is associated with chronic kidney disease and urothelial carcinomas of the upper urinary tract. Following metabolic activation, AA reacts with dA and dG residues in DNA to form aristolactam (AL)-DNA adducts. These mutagenic lesions generate a unique TP53 mutation spectrum, dominated by A:T to T:A transversions with mutations at dA residues located almost exclusively on the non-transcribed strand. We determined the level of AL-dA adducts in human fibroblasts treated with AA to determine if this marked strand bias could be accounted for by selective resistance to global-genome nucleotide excision repair (GG-NER). AL-dA adduct levels were elevated in cells deficient in GG-NER and transcription-coupled NER, but not in XPC cell lines lacking GG-NER only. In vitro, plasmids containing a single AL-dA adduct were resistant to the early recognition and incision steps of NER. Additionally, the NER damage sensor, XPC-RAD23B, failed to specifically bind to AL-DNA adducts. However, placing AL-dA in mismatched sequences promotes XPC-RAD23B binding and renders this adduct susceptible to NER, suggesting that specific structural features of this adduct prevent processing by NER. We conclude that AL-dA adducts are not recognized by GG-NER, explaining their high mutagenicity and persistence in target tissues.

Human 8-oxoguanine-DNA glycosylase (OGG1) efficiently removes mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine when paired with cytosine in oxidatively damaged DNA. Excision of 8-oxoGua mispaired with adenine may lead to G-->T transversions. Post-translational modifications such as phosphorylation could affect the cellular distribution and enzymatic activity of OGG1. Mutations and polymorphisms of OGG1 may affect the enzymatic activity and have been associated with increased risk of several cancers. In this study, we used double-stranded oligodeoxynucleotides containing 8-oxoGua:Cyt or 8-oxoGua:Ade pairs, as well as gamma-irradiated calf thymus DNA, to investigate the kinetics and substrate specificity of several known OGG1 polymorphic variants and phosphomimetic Ser-->Glu mutants. Among the polymorphic variants, A288V and S326C displayed opposite-base specificity similar to that of wild-type OGG1, whereas OGG1-D322N was 2.3-fold more specific for the correct opposite base than the wild-type enzyme. All phosphomimetic mutants displayed approximately 1.5-3-fold lower ability to remove 8-oxoGua in both assays, whereas the substrate specificity of the phosphomimetic mutants was similar to that of the wild-type enzyme. OGG1-S326C efficiently excised 8-oxoGua from oligodeoxynucleotides and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from gamma-irradiated DNA, but excised 8-oxoG rather inefficiently from gamma-irradiated DNA. Otherwise, kcat values for 8-oxoGua excision obtained from both types of experiments were similar for all OGG1 variants studied. It is known that the human AP endonuclease APEX1 can stimulate OGG1 activity by increasing its turnover rate. However, when wild-type OGG1 was replaced by one of the phosphomimetic mutants, very little stimulation of 8-oxoGua removal was observed in the presence of APEX1.

An abundant oxidative lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), often directs the misincorporation of dAMP during replication. To prevent mutations, cells possess an enzymatic system for the removal of 8-oxoG. A key element of this system is 8-oxoguanine-DNA glycosylase (Fpg in bacteria, OGG1 in eukaryotes), which must excise 8-oxoG from 8-oxoG:C pairs but not from 8-oxoG:A. We investigated the influence of various factors, including ionic strength, the presence of Mg(2+) and organic anions, polyamides, crowding agents and two small heterocyclic compounds (biotin and caffeine) on the activity and opposite-base specificity of Escherichia coli Fpg and human OGG1. The activity of both enzymes towards 8-oxoG:A decreased sharply with increasing salt and Mg(2+) concentration, whereas the activity on 8-oxoG:C was much more stable, resulting in higher opposite-base specificity when salt and Mg(2+) were at near-physiological concentrations. This tendency was observed with both Cl(-) and glutamate as the major anions in the reaction mixture. Kinetic and binding parameters for the processing of 8-oxoG:C and 8-oxoG:A by Fpg and OGG1 were determined under several different conditions. Polyamines, crowding agents, biotin and caffeine affected the activity and specificity of Fpg or OGG1 only marginally. We conclude that, in the intracellular environment, the specificity of Fpg and OGG1 for 8-oxoG:C versus 8-oxoG:A is mostly due to high ionic strength and Mg(2+).

Uracil-DNA glycosylase (Ung) can quickly locate uracil bases in an excess of undamaged DNA. DNA glycosylases may use diffusion along DNA to facilitate lesion search, resulting in processivity, the ability of glycosylases to excise closely spaced lesions without dissociating from DNA. We propose a new assay for correlated cleavage and analyze the processivity of Ung. Ung conducted correlated cleavage on double- and single-stranded substrates; the correlation declined with increasing salt concentration. Proteins in cell extracts also decreased Ung processivity. The correlated cleavage was reduced by nicks in DNA, suggesting the intact phosphodiester backbone is important for Ung processivity.