Aristolochic Acid Research Articles

Abstract LT09: Integrative Multi-Omics Enhancer Activity Profiling Identifies Therapeutic Vulnerabilities in Cholangiocarcinoma of Different Etiologies

Abstract Objectives: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. Design: Integrative multi-omics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, and patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining, and single-cell spatial transcriptomics were used to explore immunogenicity of diverse CCA. Results: We identified 3 distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumor growth in in-vitro and in-vivo models. The first group (ESTRO), with mostly fluke-associated CCAs, displayed activation in estrogen signaling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intra-tumor differences in AA mutation load were correlated to intra-tumor variation of different immune cell populations. Conclusion: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumorigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits. Citation Format: Jing Han Hong, Chern Han Yong, Hong Lee Heng, Jason Yongsheng Chan, Mai Chan Lau, Jianfeng Chen, Jing Yi Lee, Abner Herbert Lim, Zhimei Li, Peiyong Guan, Pek Lim Chu, Sheng Rong Ng, Xiaosai Yao, Peili Wang, Rong Xiao, Xian Zeng, Yichen Sun, Wei Liu, Joanna Koh, Felicia Yu Ting Wee, Jeffrey Chun Tatt Lim, Cedric Chuan Young Ng, Xiu Yi Kwek, Poramate Klanrit, Yaojun Zhang, Jiaming Lai, David Wai Meng Tai, Chawalit Pairojkul, Simona Dima, Irinel Popescu, Sen-Yung Hsieh, Ming-Chin Yu, Joe Yeong, Sarinya Kongpetch, Apinya Jusakul, Watcharin Loilome, Patrick Tan, Jing Tan, Bin Tean Teh. Integrative Multi-Omics Enhancer Activity Profiling Identifies Therapeutic Vulnerabilities in Cholangiocarcinoma of Different Etiologies [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT09.

Nephroprotective effects of coriander (Coriandrum sativum) leaves aqueous extracts in aristolochic acid-intoxicated zebrafish embryos.

Coriander is a notable medicinal plant known for its diverse properties, including anti-inflammatory, antioxidant, anticancer, analgesic, and anti-diabetic effects. Despite its recognized health benefits, research on its nephroprotective properties is limited. This study aimed to investigate the potential nephroprotective properties of an aqueous extract derived from coriander leaves using an aristolochic acid-intoxicated zebrafish model. To assess kidney abnormalities induced by aristolochic acid (AA), we utilized the transgenic line Tg(wt1b:egfp), which expresses green fluorescent protein (GFP) in the kidney. Our previous report indicated that AA exposure leads to acute renal failure in zebrafish characterized by kidney malformation and impaired renal function. However, pretreatment of coriander extract (CE) can mitigate kidney malformations induced by AA. In addition, CE pretreatment reduces the accumulation of red blood cells in the glomerular region. To verify the nephroprotective effects of CE, we analyzed renal function by measuring the glomerular filtration rate in zebrafish embryos. Results indicate that CE partially mitigates renal function impairment caused by AA exposure, suggesting its potential to attenuate AA-induced renal failure. Mechanistically, pretreatment with CE reduces the expression of proinflammatory and proapoptotic genes induced by AA. This suggests that CE likely alleviates acute renal failure by reducing inflammation and apoptosis. As a result, we regard zebrafish as a valuable model for screening natural compounds that have the potential to alleviate AA-induced nephrotoxicity.

Effects of bioconversion by Battus polydamas on the chemical composition of Aristolochia spp. and evaluation of antimicrobial activity and biocompatibility

Aristolochia plants are emblematic from an ethnopharmacological viewpoint and are know to possess numerous biological properties, including antiseptic. However, the medicinal potential of these species is debatable because of their representative chemical constituents, aristolochic acids (AAs) and aristolactams (ALs), which are associated, for instance, with nephropathy and cancer. These contrasting issues have stimulated the development of approaches intended to detoxification of aristoloquiaceous biomasses, among which is included the bioconversion method using larvae of the specialist phytophagous insect Battus polydamas, previously shown to be viable for chemical diversification and to reduce toxicity. Thus, eleven Aristolochia spp. were bioconverted, and the antimicrobial activities of the plant methanolic extracts and its respective bioconversion products were evaluated. The best results were found for Aristolochia esperanzae, Aristolochia gibertii, and Aristolochia ringens against Bacillus cereus, with MIC ranging from 7.8 to 31.25 μg/mL. These three species were selected for chemical, antioxidant, cytotoxic, hemolytic, and mutagenic analyses. Chemical analysis revealed 65 compounds, 21 of them possible bioconversion products. The extracts showed potential to inhibit the formation and degradation of B. cereus biofilms. Extracts of A. gibertii and its bioconverted biomass showed antioxidant activity comparable to dibutylhydroxytoluene (BHT) standard. Bioconversion decreased the hemolytic activity of A. esperanzae and the cytotoxicities of A. esperanzae and A. gibertii. None of the extracts was found to be mutagenic. The bioactivities of the fecal extracts were maintained, and biocompatibility was improved. Therefore, the results obtained in this study reveal positive expectations about the natural detoxification process of the Aristolochia species.

The Bacterial and Fungal Compositions in the Rhizosphere of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. in a Typical Planting Region

Asarum is a traditional Chinese medicinal plant, and its dried roots are commonly used as medicinal materials. Research into the traits of the bacteria and fungus in the Asarum rhizosphere and how they relate to the potency of medicinal plants is important. During four cropping years and collecting months, we used ITS rRNA gene amplicon and sequencing to assess the population, diversity, and predominant kinds of bacteria and fungus in the rhizosphere of Asarum. HPLC was used to determine the three bioactive ingredients, namely asarinin, aristolochic acid I, and volatile oil. The mainly secondary metabolites of Asarum, relationships between microbial communities, soil physicochemical parameters, and possible influences on microbial communities owing to various cropping years and collecting months were all statistically examined. The cropping years and collecting months affected the abundance and diversity of rhizosphere bacteria and fungi, but the cropping year had a significant impact on the structures and compositions of the bacterial communities. The rhizosphere microorganisms were influenced by both the soil physicochemical properties and enzyme activities. Additionally, this study revealed that Trichoderma was positively correlated with the three bioactive ingredients of Asarum, while Tausonia showed entirely opposite results. Gibberella and Leptosphaeria demonstrated a significantly negative correlation with asarinin and violate oil, but they were weakly correlated with the aristolochic acid I content. This study revealed variations in the Asarum rhizosphere microorganism population, diversity, and dominant types across four cropping years and collecting months. The relationship between Asarum secondary metabolites, the soil physicochemical properties, enzyme activities, and rhizosphere microorganisms was discussed. Our results will guide the exploration of the soil characteristics and rhizosphere microorganisms’ structures by regulating the microbial community to enhance Asarum quality.

Effects of Heavy Metal Co-Exposure on the formation of DNA Adducts from Aristolochic Acid I: Implications for Balkan Endemic Nephropathy Development.

Accumulated evidence has shown that Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with exposure to aristolochic acids (AA), and the associated DNA adduct formation, as a key causative factor of BEN development. Here, we show that coexposure to arsenic, cadmium, and iron increases the DNA adduct formation of AA in cultured kidney cells, while exhibiting both an exposure concentration and duration dependence. In contrast, coexposure to calcium and copper showed a decreasing DNA adduct formation. Because DNA damage is responsible for both the nephrotoxicity and carcinogenicity of AA, these results shed greater light on the endemic nature of BEN.

Aristolochic acid induces acute kidney injury through ferroptosis.

Aristolochic acid (AA)-induced acute kidney injury (AKI) presents with progressive decline in renal function and rapid progression to end-stage renal disease. Among the multiple mechanisms identified in AKI, ferroptosis has been shown to be involved in various forms of AKI. But few studies have elucidated the role of ferroptosis in AA-induced AKI. In this study, we investigated the role of ferroptosis in AA-induced acute renal tubular injury in vivo and in vitro. Mice with acute aristolochic acid nephropathy showed increased malondialdehyde levels, aggravated lipid peroxidation, decreased superoxide dismutase activity, and glutathione depletion. The expression of glutathione peroxidase 4 was decreased and the expression of acyl-CoA synthetase long-chain family member 4 was increased. Inhibition of ferroptosis by ferrostatin-1 significantly improved the renal function, reduced histopathological lesions, partially alleviated lipid peroxidation, and restored the antioxidant capacity. In vitro studies also revealed that AA significantly reduced cell viability, induced reactive oxygen species production, increased intracellular iron level and decreased ferroptosis-related protein expression. Inhibition of ferroptosis significantly increased cell viability and attenuated AA-induced renal tubular epithelial cell injury. It is suggested that ferroptosis plays an important role in AA-induced acute tubular injury. And inhibition of ferroptosis may exert renoprotective effects possibly by preventing lipid peroxidation, restoring the antioxidant activity or regulating iron metabolism.

Abstract 6160: Mutational signatures and their associations with social determinants of health

Abstract Introduction: Mutational signatures, or unique patterns of genetic mutations due to exogenous and endogenous mutational processes, are being continuously identified through the analysis of cancer patients’ genomes. While cancer research has developed and mortality rates have declined, disparities in cancer outcomes have widened. Social determinants of health (SDOH), such as access to health care, education, and economic stability are essential to addressing health equity. While much research is ongoing regarding the identification and etiology of mutational signatures, little work has been published regarding the associations of these signatures with SDOH. Therefore, this study proposes an exploration into the relationships between the presence of mutational signatures and SDOH within an adult pan-cancer patient population. Methods: Variant calls from whole exome sequencing for each patient was used to infer the presence of 58 single base substitution (SBS) mutational signatures. Descriptive statistics were calculated for 455 unique patients. Previously documented associations between primary diagnosis site and distinct signatures were used to validate the predicted signatures in the patient population. Signature profiles were then linked to 208 patient health records with complete SDOH. Logistic regression predicting the presence of each signature was modeled for these patients. Full models for each signature included age, gender, Rural-Urban Commuting Area (RUCA) code, tobacco and alcohol usage, education, income, housing condition, and access to healthcare. Stepwise selection was used to determine significant variables. Results: The most common signatures in the complete population (n = 455) were SBS1 (clock-like; 98%) and SBS39 (95%), followed by SBS5 (clock-like; 38%) and SBS22 (aristolochic acid exposure; 36%). However, SBS24 (aflatoxin exposure; p = .019) and SBS7a (UV exposure; p = .002) were significantly more common in lung and skin cancer patients respectively. Each patient had 4.1 signatures on average, with a range of 2-7. The average number of signatures per patient varied significantly by primary diagnosis (ANOVA p = .014). Patients with a vulva/vagina (4.8) or bladder/urinary tract (4.9) diagnosis had the highest number of signatures while those with a liver (3.3) or kidney (3.6) diagnosis had the lowest. Logistic regression models for the population with SDOH (n = 208) showed poor housing condition was associated with SBS4 (tobacco smoking; p = 0.091). Any issue in accessing healthcare was associated with SBS13 (AID/APOBEC; p = 0.114) and SBS21 (MMR; p = 0.076). Greater rurality was associated with SBS16 (p = .144) and SBS21 (p = .107). The relationship was inverse for SBS1 (p = .121). Conclusion: Multiple significant relationships between different SDOH and mutational signatures was revealed, pointing the need to extend this research to larger patient populations with diverse SDOH. Citation Format: Padmapriya Swaminathan, McKenna Deaton, Crystal Hattum, Benjamin Solomon, William Spanos, David Starks, Rachel Elsey, Casey Williams, Tobias Meissner. Mutational signatures and their associations with social determinants of health [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6160.

Comprehensive HRMS Screening and Risk Assessments of Aristolochic Acid Analogues in Asari Radix et Rhizoma and Related Commercial Health Products.

Aristolochic acid analogues (AAAs) are well-known toxins. We performed the first comprehensive screening on AAAs in Asari Radix et Rhizoma (underground part of Asarum heterotropoides Schmidt), the only Aristolochiaceae plant widely used in clinical practice. LC-HRMS revealed 70 trace AAAs using polygonal mass defect filtering and precursor ion list strategies, 38 of which were newly discovered in A. heterotropoides. UHPLC-QTrap-MS/MS was then utilized for quantitative/semiquantitative analysis of 26 abundant compounds. Seventeen AAAs were detected from 91 batches of A. heterotropoides and 20 AAAs from 166 consumable products. For 141 Asari-containing proprietary products, aristolactam I and aristolactam II-glucoside exhibited the widest distribution, present in 98% products. AA IVa was the most abundant, detected in 91%. Notably, 60% of the products contained AA I (0.03-0.79 ppm). The safety was assessed using linear extrapolation, permitted daily exposure, cumulative amount, and the margin of exposure. It is recommended that AA I content be limited to 3 ppm.

Ionizable copolymer functionalized magnetic nanocomposite as an adsorbent for boosting the extraction selectivity of aristolochic acids.

Aristolochic acid nephropathy (AAN) has drawn increasing public attention. Organic anion transporters (OATs) are considered to be responsible for mediating nephrotoxicity of aristolochic acids (AAs), as AAs are typical OAT1 substrates that exhibit anionic properties and contain one hydrophobic domain. Inspired by the OAT1 three-dimensional structure or substrate/protein interactions involved in transport, we designed a magnetic polymeric hybrid, mimicking the effect of basic and aromatic residues of OAT1, for efficient enriching aristolochic acid I (AA I) and aristolochic acid II (AA II) in Traditional Chinese patent medicines (TCPM). N, N-dimethylaminopropyl acrylamide (DMAPAm) was used as a cationic monomer and copolymerized with divinylbenzene (DVB) onto the surface of monodisperse magnetic nanoparticles (denoted as MNs@SiO2T-DvbDam). The magnetic polymer hybrid demonstrated high selectivity and capacity for AAs, which was mainly attributed to (1) electrostatic interactions from the cationic or basic moiety of DMAPAm and (2) the hydrophobic and π-π stacking interactions from the aromatic ring of DVB. Additionally, the surface of the hybrid exhibited amphiphilic property according to the ionization of DMAPAm, thus improving the compatibility of the adsorbent with the aqueous sample matrix. This strategy was proven to be robust in the analysis of real drug samples, which was characterized by a good linearity, high recovery and satisfactory reusability. This work confirmed that the proposed tool could be a promising candidate for enhancing the extraction selectivity of AAs in Traditional Chinese medicines (TCM).

Cognate antigen-independent differentiation of resident memory T cells in chronic kidney disease.

Resident memory T cells (TRMs), which are memory T cells that are retained locally within tissues, have recently been described as antigen-specific frontline defenders against pathogens in barrier and non-barrier epithelial tissues. They have also been noted for perpetuating chronic inflammation. The conditions responsible for TRM differentiation are still poorly understood, and their contributions, if any, to sterile models of chronic kidney disease (CKD) remain a mystery. In this study we subjected male C57BL/6J mice and OT-1 transgenic mice to five consecutive days of 2mg/kg Aristolochic acid (AA) injections i.p. to induce CKD or saline injections as a control. We evaluated their kidney immune profiles at two weeks, six weeks, and six months after treatment. We identified a substantial population of TRMs in the kidneys of mice with AA-induced CKD. Flow cytometry of injured kidneys showed T cells bearing TRM surface markers and single cell RNA sequencing revealed these cells as expressing well-known TRM transcription factors and receptors responsible for TRM differentiation and maintenance. While kidney TRMs expressed Cd44, a marker of antigen experience and T cell activation, their derivation was independent of cognate antigen-T cell receptor interactions, as the kidneys of transgenic OT-1 mice still harbored considerable proportions of TRMs after injury. Our results suggest a non-antigen-specific or antigen-independent mechanism capable of generating TRMs in the kidney and highlight the need to better understand TRMs and their involvement in CKD.

Human UDP-glucuronosyltransferase 1As catalyze aristolochic acid D O-glucuronidation to form a lesser nephrotoxic glucuronide

Ethnopharmacological relevanceAristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet. Aim of the studyTo identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity. Materials and methodsThe O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD. ResultsAAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-β-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the Km values of 4.27 μM, 9.05 μM, 3.87 μM, and 7.00 μM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction. ConclusionCollectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.

Toxicity Study on Crude Alkaloid Extracts of Houttuyniae herba Based on Zebrafish and Mice.

Houttuyniae herba has a long history of medicinal and edible homology in China. It has the functions of clearing heat and detoxifying, reducing swelling and purulent discharge, diuresis, and relieving gonorrhea. It is mainly distributed in the central, southeastern, and southwestern provinces of China. Houttuyniae herba has been designated by the National Ministry of Health of China as a dual-use plant for both food and medicine. Comprising volatile oils, flavonoids, and alkaloids as its primary constituents, Houttuyniae herba harbors aristolactams, a prominent subclass of alkaloids. Notably, the structural affinity of aristolactams to aristolochic acids is discernible, the latter known for its explicit toxicological effects. Additionally, the safety study on Houttuyniae herba mainly focused on the ethanol, methanol, or aqueous extract. In this study, both zebrafish and mice were used to evaluate the acute toxicity of the total alkaloids extracts from Houttuyniae herba (HHTAE). The zebrafish experiment showed that a high concentration (0.1 mg/mL) of HHTAE had a lethal effect on zebrafish embryos. Furthermore, the mice experiment results showed that, even at a higher dose of 2000 mg/kg, HHTAE was not toxic. In conclusion, HHTAE was of low safety risk.

Mitochondrial uptake of aristolactam I plays a critical role in its toxicity

Aristolochic acid I (AAI), a component of aristolochic acids, can be converted to the toxic metabolite Aristolactam I (ALI) in vivo which forms aristolactam-nitrenium with delocalized positive charges. It is widely accepted that delocalized lipophilic cations can accumulate in mitochondria due to the highly negatively charged microenvironment of the mitochondrial matrix, but the uptake of ALI by mitochondria is not known. In this study, the cell uptake and mitochondrial localization of ALI, and its subsequent impact on mitochondrial function were investigated. Results show that ALI can rapidly penetrate HK-2 cells without relying on organic anion transporters 1/3 (OAT1/3). The cellular distribution of ALI was found to align with the observed distribution of a mitochondria-selective dye in HK-2 cells. Furthermore, the cell uptake and mitochondrial uptake of ALI were both inhibited by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, which induces mitochondrial membrane depolarization. These results suggest that ALI is selectively taken up by mitochondria. Consequently, mitochondrial dysfunction was observed after treatment with ALI. It should be noted that inhibiting OAT1/3 could result in an increased exposure of ALI in vivo and cause more seriously nephrotoxicity. In conclusion, this research reports the mitochondrial uptake of ALI and provides new insight on potential strategies for protection against AAI-induced nephrotoxicity.

Tubular CPT1A deletion minimally affects aging and chronic kidney injury.

Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal β-oxidation when mitochondrial FAO is impaired.

Cholangiocarcinoma: Recent Advances in Molecular Pathobiology and Therapeutic Approaches.

Cholangiocarcinomas (CCA) pose a complex challenge in oncology due to diverse etiologies, necessitating tailored therapeutic approaches. This review discusses the risk factors, molecular pathology, and current therapeutic options for CCA and explores the emerging strategies encompassing targeted therapies, immunotherapy, novel compounds from natural sources, and modulation of gut microbiota. CCA are driven by an intricate landscape of genetic mutations, epigenetic dysregulation, and post-transcriptional modification, which differs based on geography (e.g., for liver fluke versus non-liver fluke-driven CCA) and exposure to environmental carcinogens (e.g., exposure to aristolochic acid). Liquid biopsy, including circulating cell-free DNA, is a potential diagnostic tool for CCA, which warrants further investigations. Currently, surgical resection is the primary curative treatment for CCA despite the technical challenges. Adjuvant chemotherapy, including cisplatin and gemcitabine, is standard for advanced, unresectable, or recurrent CCA. Second-line therapy options, such as FOLFOX (oxaliplatin and 5-FU), and the significance of radiation therapy in adjuvant, neoadjuvant, and palliative settings are also discussed. This review underscores the need for personalized therapies and demonstrates the shift towards precision medicine in CCA treatment. The development of targeted therapies, including FDA-approved drugs inhibiting FGFR2 gene fusions and IDH1 mutations, is of major research focus. Investigations into immune checkpoint inhibitors have also revealed potential clinical benefits, although improvements in survival remain elusive, especially across patient demographics. Novel compounds from natural sources exhibit anti-CCA activity, while microbiota dysbiosis emerges as a potential contributor to CCA progression, necessitating further exploration of their direct impact and mechanisms through in-depth research and clinical studies. In the future, extensive translational research efforts are imperative to bridge existing gaps and optimize therapeutic strategies to improve therapeutic outcomes for this complex malignancy.

Application of duplex sequencing to evaluate mutagenicity of aristolochic acid and methapyrilene in Fisher 344 rats

Duplex sequencing (DS) is an error-corrected next-generation sequencing (NGS) method that can overcome notorious high error rate from the process of NGS and detect ultralow-frequency mutations. In this study, we evaluated the mutagenicity of aristolochic acid, a known genotoxic carcinogen, and methapyrilene, a known nongenotoxic carcinogen using DS. Four male Fisher 344 rats were treated with aristolochic acid, methapyrilene, or the vehicle control for 6 weeks, liver tissues were collected one day after the treatment, and the DNA was isolated for analysis. The mutation frequency for the aristolochic acid-treated group was significantly increased over the vehicle control (44-fold), whereas no significant difference in the mutation frequency was observed between the methapyrilene-treated and the control groups. The primary type of mutation induced by aristolochic acid was A:T > T:A transversion, which occurred frequently at ApT sites, whereas the major type of mutation in the control and methapyrilene-treated groups was G:C > A:T transition, which occurred frequently at CpG sites. These findings are consistent with previously published data obtained with other in vivo mutation assays. Thus, our results suggest that the DS mutation assay is a promising technology for assessing mutagenicity of chemicals in vivo.

PSTPIP2 ameliorates aristolochic acid nephropathy by suppressing interleukin-19-mediated neutrophil extracellular trap formation.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by herbal medicines. Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) and neutrophil extracellular traps (NETs) play important roles in kidney injury and immune defense, respectively, but the mechanism underlying AAN regulation by PSTPIP2 and NETs remains unclear. We found that renal tubular epithelial cell (RTEC) apoptosis, neutrophil infiltration, inflammatory factor, and NET production were increased in a mouse model of AAN, while PSTPIP2 expression was low. Conditional knock-in of Pstpip2 in mouse kidneys inhibited cell apoptosis, reduced neutrophil infiltration, suppressed the production of inflammatory factors and NETs, and ameliorated renal dysfunction. Conversely, downregulation of Pstpip2 expression promoted kidney injury. In vivo, the use of Ly6G-neutralizing antibody to remove neutrophils and peptidyl arginine deiminase 4 (PAD4) inhibitors to prevent NET formation reduced apoptosis, alleviating kidney injury. In vitro, damaged RTECs released interleukin-19 (IL-19) via the PSTPIP2/nuclear factor (NF)-κB pathway and induced NET formation via the IL-20Rβ receptor. Concurrently, NETs promoted apoptosis of damaged RTECs. PSTPIP2 affected NET formation by regulating IL-19 expression via inhibition of NF-κB pathway activation in RTECs, inhibiting RTEC apoptosis, and reducing kidney damage. Our findings indicated that neutrophils and NETs play a key role in AAN and therapeutic targeting of PSTPIP2/NF-κB/IL-19/IL-20Rβ might extend novel strategies to minimize Aristolochic acid I-mediated acute kidney injury and apoptosis.

Mutational signature and prognosis in adenocarcinoma of bladder.

655 Background: Adenocarcinoma of bladder is a rare urinary bladder carcinoma with limited therapy options due to lack of molecular characterization. Here we aim to reveal mutational and transcriptomic landscapes of adenocarcinoma of bladder and their relationship with prognosis. Methods: Between February 2015 and June 2021, a total of 23 patients with adenocarcinoma of bladder were included. These included 16 patients with primary bladder adenocarcinomas and 7 patients with urachal adenocarcinoma. Whole exome sequencing, whole genome sequencing, bulk RNA-Seq and single-cell RNA-Seq were conducted for the specimens. Correlation analysis, survival analysis and t tests were also performed. Results: Prevalent T>A substitutions were observed among somatic mutations, and major tri-nucleotide contexts included 5′-CTC-3′and 5′-CTG-3′. This pattern was mainly contributed by COSMIC Signature 22 related to chemical carcinogen exposure (probably aristolochic acid), which has not been reported in bladder adenocarcinoma. Moreover, genes with copy number changes also enriched KEGG term “chemical carcinogenesis”. Transcriptomic analysis implied high immune infiltration and luminal-like feature in majority of samples. Interestingly, a small fraction of samples with APOBEC-derived mutational signature exhibited higher risk of disease progression compared with samples with only chemical carcinogen-related signature, confirming molecular and prognosis heterogeneity in bladder adenocarcinoma. Conclusions: This study presents mutational and transcriptomic landscapes of bladder adenocarcinoma, and chemical carcinogen-related mutational signature indicates good prognosis in adenocarcinoma of bladder.

Histone deacetylase-mediated silencing of PSTPIP2 expression contributes to aristolochic acid nephropathy-induced PANoptosis.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by using herbal medicines. Currently, no therapies are available to treat or prevent aristolochic acid nephropathy. Histone deacetylase (HDAC) plays a crucial role in the development and progression of renal disease. We tested whether HDAC inhibitors could prevent aristolochic acid nephropathy and determined the underlying mechanism. HDACs expression in the aristolochic acid nephropathy model was examined. The activation of PANoptosis of mouse kidney and renal tubular epithelial cell were assessed after exposure to HDAC1 and HDAC2 blockade. Kidney-specific knock-in of proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) mice were used to investigate whether PSTPIP2 affected the production of PANoptosome. Aristolochic acid upregulated the expression of HDAC1 and HDAC2 in the kidneys. Notably, the HDAC1 and HDAC2 specific inhibitor, romidepsin (FK228, depsipeptide), suppressed aristolochic acid-induced kidney injury, epithelial cell pyroptosis, apoptosis and necroptosis (PANoptosis). Moreover, romidepsin upregulated PSTPIP2 in renal tubular epithelial cells, which was enhanced by aristolochic acid treatment. Conditional knock-in of PSTPIP2 in the kidney protected against aristolochic acid nephropathy. In contrast, the knockdown of PSTPIP2 expression in PSTPIP2-knock-in mice restored kidney damage and PANoptosis. PSTPIP2 function was determined in vitro using PSTPIP2 knockdown or overexpression in mouse renal tubular epithelial cells (mTECs). Additionally, PSTPIP2 was found to regulate caspase 8 in aristolochic acid nephropathy. HDAC-mediated silencing of PSTPIP2 may contribute to aristolochic acid nephropathy. Hence, HDAC1 and HDAC2 specific inhibitors or PSTPIP2 could be valuable therapeutic agents for preventing aristolochic acid nephropathy.

Development and Application of a Comprehensive Nontargeted Screening Strategy for Aristolochic Acid Analogues.

Aristolochic acid analogs (AAAs) are naturally occurring carcinogenic and toxic compounds that pose a safety threat to pharmaceuticals and the environment. It is challenging to screen AAAs due to their lack of characteristic mass spectral fragmentation and their presence of structural diversity. A comprehensive nontargeted screening strategy was proposed by taking into account diverse factors and incorporating various self-developed techniques, and a Python3-based toolkit called AAAs_finder was developed for its implementation. The main procedures consist of virtual structure and ultraviolet and visible (UV) spectra database creation, exact mass and UV spectra-based suspect data extraction, tandem mass spectra (MS/MS) anthropomorphic interpretation, and multicondition retention time (RT) prediction-based candidate structures ranking. To initially assess screening feasibility, eight hypothetical unknown samples were subjected to nontargeted screening using the AAAs_finder toolkit and two other advanced tools. The results showed that the former successfully identified all, while the latter two only managed to identify two and three, respectively, indicating that our strategy was more feasible. After that, the strategy was carefully evaluated for false positives and false negatives, instrument dependence, reproducibility, and sensitivity. After the evaluation, the strategy was successfully applied to the screening of AAAs in real samples, such as herbal medicine, spiked soil, and water. Overall, this study proposed a nontargeted screening strategy and toolkit independent of characteristic mass spectral fragmentation and able to overcome challenges posed by structural diversity for the AAAs screening, which is also valuable for other classes of compounds.