Asunto(s)

Ácidos Aristolóquicos , Medicamentos Herbarios Chinos , Humanos , Ácidos Aristolóquicos/efectos adversos , Masculino , Lactante , Medicamentos Herbarios Chinos/efectos adversos , Enfermedades Renales/inducido químicamente , Aristolochia/química , Aristolochia/efectos adversos , Riñón/patología , Riñón/efectos de los fármacos

RESUMEN

Aristolochic acid nephropathy (AAN) is a rapidly progressive kidney disease caused by medical or environmental exposure to aristolochic acids (AAs). This study aimed to identify serum metabolites associated with the severity of acute AAN and investigate the underlying mechanisms. Male C57BL/6 mice were treated with vehicle and 3 doses of aristolochic acid I (AAI) (1.25, 2.5, and 5 mg/kg/d) for 5 days by intraperitoneal injection. The results showed that AAI dose-dependently increased blood urea nitrogen (BUN) and serum creatinine (Scr) levels and renal pathological damage. Non-targeted metabolomics revealed that differences in serum metabolite profiles from controls increased with increasing AAI doses. Compared with the control group, 56 differentially expressed metabolites (DEMs) that could be affected by all 3 doses of AAI were obtained. We further identified 13 DEMs whose abundance significantly correlated with Scr and BUN levels and had good predictive values for diagnosing AAI exposure. Among the 13 DEMs, lipids and lipid-like molecules constituted the majority. Western blotting found that AAI suppressed renal fatty acid oxidation (FAO)-related enzymes expression. In conclusion, these findings provided evidence for developing biomarkers for monitoring AAs exposure and AAN diagnosis and indicated activation of FAO as a potential direction for the treatment of AAN.

Asunto(s)

Ácidos Aristolóquicos , Biomarcadores , Ratones Endogámicos C57BL , Ácidos Aristolóquicos/toxicidad , Animales , Masculino , Biomarcadores/sangre , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/inducido químicamente , Enfermedades Renales/sangre , Enfermedades Renales/patología , Nitrógeno de la Urea Sanguínea , Metabolómica , Ratones , Creatinina/sangre , Relación Dosis-Respuesta a Droga , Índice de Severidad de la Enfermedad , Modelos Animales de Enfermedad

RESUMEN

Collecting duct carcinoma (CDC) is an aggressive rare subtype of kidney cancer with unmet clinical needs. Little is known about its underlying molecular alterations and etiology, primarily due to its rarity, and lack of preclinical models. This study aims to comprehensively characterize molecular alterations in CDC and identify its therapeutic vulnerabilities. Through whole-exome and transcriptome sequencing, we identified KRAS hotspot mutations (G12A/D/V) in 3/13 (23%) of the patients, in addition to known TP53, NF2 mutations. 3/13 (23%) patients carried a mutational signature (SBS22) caused by aristolochic acid (AA) exposures, known to be more prevalent in Asia, highlighting a geologically specific disease etiology. We further discovered that cell cycle-related pathways were the most predominantly dysregulated pathways. Our drug screening with our newly established CDC preclinical models identified a CDK9 inhibitor LDC000067 that specifically inhibited CDC tumor growth and prolonged survival. Our study not only improved our understanding of oncogenic molecular alterations of Asian CDC, but also identified cell-cycle machinery as a therapeutic vulnerability, laying the foundation for clinical trials to treat patients with such aggressive cancer.

Asunto(s)

Carcinoma de Células Renales , Neoplasias Renales , Humanos , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Neoplasias Renales/genética , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Femenino , Ratones , Mutación , Masculino , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ácidos Aristolóquicos/farmacología , Persona de Mediana Edad , Línea Celular Tumoral , Secuenciación del Exoma , Anciano , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico

RESUMEN

Aristolochic acids are one of the major compounds in aristolochia plants, which are nephrotoxic and carcinogenic. A method was established for the detection and identification of aristolochic acids and their DNA adducts in four different herbs using ultra-high performance liquid chromatography-ion mobility quadrupole time-of flight mass spectrometry. Solid phase extraction conditions were optimized to improve the sensitivity of the experiment by using 40 mg of C18 as adsorbent and 100 µL ethanol as elution solvent. At a collision energy of 10-40 eV, these compounds and cleavage patterns were precisely identified and analyzed by secondary fragmentation and collision cross section values. The obtained mass spectrometry data were then analyzed by targeted metabolomics, including principal component analysis, partial least squares-discriminant analysis and hierarchical clustering analysis, and importing the samples in the established model, the confidence values can reach 0.61 and 0.76. All in all, this method can provide a useful tool for the detection of aristolochic acids and deoxyribonucleic acid adducts. In conclusion, this method was successfully used for the detection and identification of aristolochic acids and their DNA adducts.

Asunto(s)

Aristolochia , Ácidos Aristolóquicos , Aductos de ADN , Metabolómica , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/análisis , Aductos de ADN/análisis , Aductos de ADN/química , Cromatografía Líquida de Alta Presión/métodos , Aristolochia/química , Metabolómica/métodos , Espectrometría de Masas/métodos , Extracción en Fase Sólida , Análisis de Componente Principal , Espectrometría de Movilidad Iónica/métodos

RESUMEN

This study presents the development and validation of an innovative microfluidic liver-on-a-chip device utilizing gravity-driven perfusion for the evaluation of drug hepatotoxicity. This research involved the construction of a hydrogel-based coculture chip that integrates liver parenchymal and stellate cells within a tri-channel configuration. The assembly and operation of the liver-on-a-chip and its accompanying custom rocker were straightforward. The cells in the chip maintained high viability and continuously synthesized liver albumin over extended culture durations. Acetaminophen (APAP), a hepatic injury-inducing drug, was utilized as a positive control in hepatic toxicity assays on the chip. The liver chip exhibited hepatotoxic responses comparable to those observed in 2D models. Furthermore, in this study we evaluated the effects of two plant-derived natural compounds, aristolochic acid I (AA) and its analog aristolactam AII (AL), in both 2D cell models and the liver-on-a-chip system. AA, known for its hepatorenal toxicity, was observed to cause hepatotoxicity in both the 2D models and on the chip. The flow cytometry and mRNA sequencing results confirmed the propensity of these compounds to induce liver cell apoptosis. Notably, AL, previously considered nontoxic, provoked a significant decrease in the hepatic functionality marker albumin exclusively in the liver chip but not in 2D models, indicating the liver chip's enhanced sensitivity to toxic substances. In summary, this pumpless liver-on-a-chip is a simple yet powerful tool for drug hepatotoxicity studies.

Asunto(s)

Acetaminofén , Dispositivos Laboratorio en un Chip , Hígado , Humanos , Acetaminofén/toxicidad , Hígado/efectos de los fármacos , Hígado/patología , Ácidos Aristolóquicos/toxicidad , Apoptosis/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Supervivencia Celular/efectos de los fármacos , Células Hep G2 , Técnicas de Cocultivo/métodos

RESUMEN

Balkan endemic nephropathy (BEN) is a chronic kidney disease that predominantly affects inhabitants of rural farming communities along the Danube River tributaries in the Balkans. Long-standing research has identified dietary exposure to aristolochic acids (AAs) as the principal toxicological cause. This study investigates the pathophysiological role of anemia in BEN, noting its earlier and more severe manifestation in BEN patients compared to those with other chronic kidney diseases. Utilizing a mouse model, our research demonstrates that prolonged exposure to aristolochic acid I (AA-I) (the most prevalent AA variant) leads to significant red blood cell depletion through DNA damage, such as DNA adduct formation in bone marrow, prior to observable kidney function decline. Furthermore, in vitro experiments with kidney cells exposed to lowered oxygen and pH conditions mimicking an anemia environment show enhanced DNA adduct formation, suggesting increased AA-I mutagenicity and carcinogenicity. These findings indicate for the first time a positive feedback mechanism of AA-induced anemia, DNA damage, and kidney impairment in BEN progression. These results not only advance our understanding of the underlying mechanisms of BEN but also highlight anemia as a potential target for early BEN diagnosis and therapy.

Asunto(s)

Anemia , Ácidos Aristolóquicos , Nefropatía de los Balcanes , Aductos de ADN , Ácidos Aristolóquicos/toxicidad , Ácidos Aristolóquicos/efectos adversos , Nefropatía de los Balcanes/inducido químicamente , Nefropatía de los Balcanes/metabolismo , Nefropatía de los Balcanes/genética , Aductos de ADN/metabolismo , Animales , Ratones , Humanos , Anemia/inducido químicamente , Anemia/metabolismo , Anemia/genética , Masculino , Daño del ADN/efectos de los fármacos , Ratones Endogámicos C57BL , Riñón/efectos de los fármacos , Riñón/metabolismo , Femenino

RESUMEN

Kidney disease represents a major medical and economic burden for which improved treatments are urgently needed. Emerging data have implicated Th17 cells and IL-17 signaling in the underlying pathogenesis of autoantibody-induced glomerulonephritis (AGN). However, the downstream transduction pathways mediated by IL-17 in autoimmunity are not well defined. In this article, we show that CCAAT/enhancer-binding protein (C/EBP) δ is elevated in kidney biopsies from multiple manifestations of human AGN. C/EBPδ is similarly upregulated in a mouse model of anti-glomerular basement membrane protein-mediated kidney disease, and Cebpd-/- mice were fully refractory to disease. Although C/EBPδ is expressed in a variety of cell types, C/EBPδ was required only in the radioresistant compartment to drive GN pathology. C/EBPδ induced expression of several IL-17-induced kidney injury markers and cytokines implicated in disease, including Il6 and Lcn2. Because mouse AGN models do not progress to fibrosis, we employed a nephrotoxic injury model using aristolochic acid I to assess the contribution of the IL-17-C/EBPδ pathway to renal fibrotic events. Surprisingly, deficiency of either C/EBPδ or the IL-17 receptor caused kidney fibrosis to be enhanced. Thus, C/EBPδ and IL-17 play divergent and apparently stage-specific roles in the pathogenesis of kidney disease.

Asunto(s)

Proteína delta de Unión al Potenciador CCAAT , Glomerulonefritis , Animales , Humanos , Ratones , Ácidos Aristolóquicos/toxicidad , Autoanticuerpos/inmunología , Proteína delta de Unión al Potenciador CCAAT/genética , Modelos Animales de Enfermedad , Glomerulonefritis/inmunología , Glomerulonefritis/patología , Interleucina-17/inmunología , Interleucina-17/metabolismo , Riñón/inmunología , Riñón/patología , Lipocalina 2/genética , Lipocalina 2/metabolismo , Lipocalina 2/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Células Th17/inmunología

RESUMEN

The sense of taste is essential for survival, as it allows animals to distinguish between foods that are nutritious from those that are toxic. However, innate responses to different tastants can be modulated or even reversed under pathological conditions. Here, we examined whether and how the internal status of an animal impacts taste valence by using Drosophila models of hyperproliferation in the gut. In all three models where we expressed proliferation-inducing transgenes in intestinal stem cells (ISCs), hyperproliferation of ISCs caused a tumor-like phenotype in the gut. While tumor-bearing flies had no deficiency in overall food intake, strikingly, they exhibited an increased gustatory preference for aristolochic acid (ARI), which is a bitter and normally aversive plant-derived chemical. ARI had anti-tumor effects in all three of our gut hyperproliferation models. For other aversive chemicals we tested that are bitter but do not have anti-tumor effects, gut tumors did not affect avoidance behaviors. We demonstrated that bitter-sensing gustatory receptor neurons (GRNs) in tumor-bearing flies respond normally to ARI. Therefore, the internal pathology of gut hyperproliferation affects neural circuits that determine taste valence postsynaptic to GRNs rather than altering taste identity by GRNs. Overall, our data suggest that increased consumption of ARI may represent an attempt at self-medication. Finally, although ARI's potential use as a chemotherapeutic agent is limited by its known toxicity in the liver and kidney, our findings suggest that tumor-bearing flies might be a useful animal model to screen for novel anti-tumor drugs.

Asunto(s)

Drosophila melanogaster , Gusto , Animales , Gusto/fisiología , Drosophila melanogaster/fisiología , Drosophila melanogaster/efectos de los fármacos , Ácidos Aristolóquicos , Neoplasias Intestinales/tratamiento farmacológico , Neoplasias Intestinales/patología

RESUMEN

Tens of thousands of people in southern Europe suffer from Balkan endemic nephropathy (BEN), and four times as many are at risk. Incidental ingestion of aristolochic acids (AAs), stemming from the ubiquitousAristolochia clematitis(birthwort) weed in the region, leads to DNA adduct-induced toxicity in kidney cells, the primary cause of BEN. Numerous cofactors, including toxic organics and metals, have been investigated, but all have shown small contributions to the overall BEN relative to non-BEN village distribution gradients. Here, we reveal that combustion-derived pollutants from wood and coal burning in Serbia also contaminate arable soil and test as plausible causative factors of BEN. Using a GC-MS screening method, biomass-burning-derived furfural and coal-burning-derived medium-chain alkanes were detected in soil samples from BEN endemic areas levels at up to 63-times and 14-times higher, respectively, than in nonendemic areas. Significantly higher amounts were also detected in colocated wheat grains. Coexposure studies with cultured kidney cells showed that these pollutants enhance DNA adduct formation by AA, - the cause of AA nephrotoxicity and carcinogenicity. With the coincidence of birthwort-derived AAs and the widespread practice of biomass and coal burning for household cooking and heating purposes and agricultural burning in rural low-lying flood-affected areas in the Balkans, these results implicate combustion-derived pollutants in promoting the development of BEN.

Asunto(s)

Nefropatía de los Balcanes , Inundaciones , Nefropatía de los Balcanes/inducido químicamente , Nefropatía de los Balcanes/epidemiología , Humanos , Carbón Mineral , Serbia , Contaminantes del Suelo/toxicidad , Ácidos Aristolóquicos , Animales , Aristolochia/química , Peninsula Balcánica , Madera , Enfermedades Renales/inducido químicamente

RESUMEN

Aristolochic acids (AAs), which are a group of nitrophenanthrene carboxylic acids formed by Aristolochia plant, have become an increasing serious threat to humans due to their nephrotoxicity and carcinogenicity. Fast and accurate approaches capable of simultaneous sensing of aristolochic acids (I-IV) are vital to avoid intake of such compounds. In this research, the novel ratiometric fluorescence zinc metal-organic framework and its nanowire have been prepared. The two different coordination modes (tetrahedral configuration and twisted triangular bipyramidal configuration) within zinc metal-organic framework lead to the significant double emissions. The ratiometric fluorescence approach based on nanowire provides a broader concentration range (3.00 × 10-7~1.00 × 10-4 M) and lower limit of detection (3.70 × 10-8 M) than that based on zinc metal-organic framework (1.00 × 10-6~1.00 × 10-4 M, 5.91 × 10-7 M). The RSDs of the results are in the range 1.4-3.5% (nanowire). The density functional theory calculations and UV-Vis absorption verify that the sensing mechanism is due to charge transfer and energy transfer. Excellent spiked recoveries for AAs(I-IV) in soil and water support that nanowire is competent to simultaneously detect these targets in real samples, and the proposed approach has potential as a fluorescence sensing platform for the simultaneous detection of AAs (I-IV) in complex systems.

Asunto(s)

Ácidos Aristolóquicos , Límite de Detección , Estructuras Metalorgánicas , Nanocables , Ácidos Aristolóquicos/análisis , Ácidos Aristolóquicos/química , Estructuras Metalorgánicas/química , Nanocables/química , Zinc/química , Espectrometría de Fluorescencia/métodos , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/química , Mediciones Luminiscentes/métodos , Colorantes Fluorescentes/química

RESUMEN

International differences in the incidence of many cancer types indicate the existence of carcinogen exposures that have not yet been identified by conventional epidemiology make a substantial contribution to cancer burden1. In clear cell renal cell carcinoma, obesity, hypertension and tobacco smoking are risk factors, but they do not explain the geographical variation in its incidence2. Underlying causes can be inferred by sequencing the genomes of cancers from populations with different incidence rates and detecting differences in patterns of somatic mutations. Here we sequenced 962 clear cell renal cell carcinomas from 11 countries with varying incidence. The somatic mutation profiles differed between countries. In Romania, Serbia and Thailand, mutational signatures characteristic of aristolochic acid compounds were present in most cases, but these were rare elsewhere. In Japan, a mutational signature of unknown cause was found in more than 70% of cases but in less than 2% elsewhere. A further mutational signature of unknown cause was ubiquitous but exhibited higher mutation loads in countries with higher incidence rates of kidney cancer. Known signatures of tobacco smoking correlated with tobacco consumption, but no signature was associated with obesity or hypertension, suggesting that non-mutagenic mechanisms of action underlie these risk factors. The results of this study indicate the existence of multiple, geographically variable, mutagenic exposures that potentially affect tens of millions of people and illustrate the opportunities for new insights into cancer causation through large-scale global cancer genomics.

Asunto(s)

Carcinoma de Células Renales , Exposición a Riesgos Ambientales , Geografía , Neoplasias Renales , Mutágenos , Mutación , Femenino , Humanos , Masculino , Ácidos Aristolóquicos/efectos adversos , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/epidemiología , Carcinoma de Células Renales/inducido químicamente , Exposición a Riesgos Ambientales/efectos adversos , Exposición a Riesgos Ambientales/análisis , Genoma Humano/genética , Genómica , Hipertensión/epidemiología , Incidencia , Japón/epidemiología , Neoplasias Renales/genética , Neoplasias Renales/epidemiología , Neoplasias Renales/inducido químicamente , Mutágenos/efectos adversos , Obesidad/epidemiología , Factores de Riesgo , Rumanía/epidemiología , Serbia/epidemiología , Tailandia/epidemiología , Fumar Tabaco/efectos adversos , Fumar Tabaco/genética

RESUMEN

Aristolochic acids (AAs) naturally occurring in the herbal genus Aristolochia are associated with a high risk of kidney failure, multiple tumors and cancers. However, approaches with high selectivity and rapidity for measuring AAs in biological samples are still inadequate. Inspired by the mechanism of AAs-induced nephrotoxicity, we designed a hybrid magnetic polymer-porous agarose (denoted as MNs@SiO2M@DNV-A), mimicking the effect of basic and aromatic residues of organic anion transporter 1 (OAT1) for efficient enriching aristolochic acid I (AA I) and aristolochic acid II (AA II) in the plasma. The monomers of vinylbenzyl trimethylammonium chloride (VBTAC), N-vinyl-2-pyrrolidinone (NVP) and divinylbenzene (DVB) were employed to construct the polymer layer, which provided a selective adsorption for AAs by multiple interactions. The porous agarose shell contributed to remove interfering proteins in the plasma samples. A magnetic solid-phase extraction (MSPE) based on the proposed composite enhanced the selectivity toward AA I and AA II in the plasma samples. In combination of HPLC analysis, the proposed method was proved to be applicable to fast and specific quantification of AAs in blood samples, which was characterized by a good linearity, high sensitivity, acceptable recovery, excellent repeatability and satisfactory reusability.

Asunto(s)

Ácidos Aristolóquicos , Compuestos de Amonio Cuaternario , Sefarosa , Extracción en Fase Sólida , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/aislamiento & purificación , Ácidos Aristolóquicos/sangre , Sefarosa/química , Extracción en Fase Sólida/métodos , Compuestos de Amonio Cuaternario/química , Cromatografía Líquida de Alta Presión/métodos , Porosidad , Límite de Detección , Animales , Humanos , Polímeros/química , Adsorción , Reproducibilidad de los Resultados

RESUMEN

In recent years, the nephrotoxicity and carcinogenicity of aristolochic acid have attracted worldwide attention, and the traditional Chinese medicine containing this ingredient has been banned in many places, affecting the TCM industry. To meet this challenge, researchers have developed various detection methods, such as high-performance liquid chromatography, gas chromatography-mass spectrometry and thin-layer chromatography. A rapid detection method must therefore be developed to ensure safety. A polyclonal antibody capable of recognizing aristolochic acid was prepared, and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established to detect the amount of aristolochic acid in the sample to be measured. Methods Using 1-(4-chlorophenyl) cyclobutylamine as a hapten, immunogens and coating antigens were obtained by coupling with bovine serum albumin (BSA) and chicken ovalbumin (OVA) using the active ester method. UV scanning confirmed the successful coupling of the conjugate, and New Zealand white rabbits were immunized. The obtained antibody serum was screened for the best antibody by ic-ELISA detection. Use the chessboard method to determine three optimal combinations of original coating concentration and antibody dilution ratio, establish a standard curve for each combination to obtain the best combination, and establish a rapid detection method. Finally, the standard aristolochic acid A was added to the purchased apple vinegar and canned coffee for recycling experiments to verify the detection method.By changing the antigen antibody concentration, the antibody showed the highest sensitivity to aristolochic acid standard at the original coating, 1000-fold dilution, IC50 of 24.88 ng/mL, limit of detection IC10 of 3.19 ng/mL, and detection range IC20-IC80 of 6.81-90.91 ng/mL. The recovery experiments under this conditions yielded a recovery rate of 92%-105%, within reasonable limits, indicating the success of the ELISA rapid detection method. Conclusion The enzyme-linked immunoassay method established in this paper can quickly detect the content of aristolochic acid in the sample to be tested, and the antibody prepared by this method has good broad-spectrum and can detect other aristolochic acid, such as aristolochic acid A, aristolochic acid B, aristolochic acid C, and aristolochic acid D.

Asunto(s)

Ácidos Aristolóquicos , Ensayo de Inmunoadsorción Enzimática , Ácidos Aristolóquicos/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Animales , Conejos , Anticuerpos , Haptenos

RESUMEN

Emerging evidence showing urothelial cancer in herbalists is linked to aristolochic acid (AA) exposure; however, the exposure pathway remains unclear. Here, we show that dermal contact and inhalation of fine powders of AA-containing herbs are significant occupational AA exposure pathways for herbalists. We initiated the study by quantifying the amount of AA in the AA-containing powder deposited on gloves and face masks worn by the operators of an AA-containing herb grinding machine. Then, we measured the kinetics of dermal absorption and dissolution of AA from fine powders of AA-containing herbs into artificial sweat and surrogate lung fluid. Lastly, we quantified the mutagenic AA-DNA adduct levels formed in the kidneys of mice exposed to AA-containing fine powders through dermal contact. Our findings highlight an urgent occupational risk that should demand implementation of safety standards for herbalists exposed to AA-containing fine powders.

Asunto(s)

Ácidos Aristolóquicos , Exposición Profesional , Polvos , Ácidos Aristolóquicos/análisis , Exposición Profesional/efectos adversos , Polvos/química , Animales , Humanos , Ratones , Aductos de ADN/análisis , Exposición por Inhalación/efectos adversos , Urotelio/efectos de los fármacos , Urotelio/patología , Practicantes de la Medicina Tradicional

RESUMEN

Early detection of drug-induced kidney injury is essential for drug development. In this study, multiple low-dose aristolochic acid (AA) and cisplatin (Cis) injections increased renal mRNA levels of inflammation, fibrosis, and renal tubule injury markers. We applied a serum amyloid A3 (Saa3) promoter-driven luciferase reporter (Saa3 promoter-luc mice) to these two tubulointerstitial nephritis models and performed in vivo bioluminescence imaging to monitor early renal pathologies. The bioluminescent signals from renal tissues with AA or CIS injections were stronger than those from normal kidney tissues obtained from normal mice. To verify whether the visualized bioluminescence signal was specifically generated by the injured kidney, we performed in vivo bioluminescence analysis after opening the stomachs of Saa3 promoter-luc mice, and the Saa3-mediated bioluminescent signal was specifically detected in the injured kidney. This study showed that Saa3 promoter activity is a potent non-invasive indicator for the early detection of drug-induced nephrotoxicity.

Asunto(s)

Ácidos Aristolóquicos , Luciferasas , Regiones Promotoras Genéticas , Proteína Amiloide A Sérica , Animales , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Ratones , Luciferasas/metabolismo , Luciferasas/genética , Ácidos Aristolóquicos/toxicidad , Genes Reporteros , Cisplatino/toxicidad , Cisplatino/efectos adversos , Mediciones Luminiscentes/métodos , Masculino , Enfermedades Renales/inducido químicamente , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Riñón/metabolismo , Riñón/efectos de los fármacos , Riñón/patología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Kelisha capsules (KLS) are often used to treat acute diarrhoea, bacillary dysentery, heat stroke, and other diseases. One of its components, Asarum, contains aristolochic acid I which is both nephrotoxic and carcinogenic. However, the aristolochic acid (AA) content in KLS and its toxicity remain unclear. AIM OF THE STUDY: The aims of this study were to quantitatively determine the contents of five aristolochic acid analogues (AAAs) in Asarum and KLS, and systematically evaluate the in vivo toxicity of KLS in rats. MATERIALS AND METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine the content of the five AAAs in Asarum and KLS. Sprague-Dawley rats were administered KLS at 0, 0.75, 1.5, and 3.0 g/kg respectively, and then sacrificed after 4 weeks of administration or after an additional 2 weeks of recovery. The endpoints assessed included body weight measurements, serum biochemistry and haematology indices, and clinical and histopathological observations. RESULTS: The AAAs content in Asarum sieboldii Miq. (HB-ESBJ) were much lower than those of the other Asarums. The contents of AA I, AA IVa, and aristolactam I in KLS were in the ranges of 0.03-0.06 µg/g, 1.89-2.16 µg/g, and 0.55-1.60 µg/g, respectively, whereas AA II and AA IIIa were not detected. None of the rats showed symptoms of toxic reactions and KLS was well tolerated throughout the study. Compared to the control group, the activated partial thromboplastin time values of rats in the 1.5 and 3.0 g/kg groups significantly reduced after administration (P < 0.05). In addition, the serum triglycerides of male rats in the 0.75 and 1.5 g/kg groups after administration, and the 0.75, 1.5, 3.0 g/kg groups after recovery were significantly decreased (P < 0.01 or P < 0.001). No significant drug-related toxicological changes were observed in other serum biochemical indices, haematology, or histopathology. CONCLUSIONS: The AA I content in KLS met the limit requirements (<0.001%) of the Chinese Pharmacopoeia. Therefore, it is safe to use KLS in the short-term. However, for safety considerations, attention should be paid to the effects of long-term KLS administration on coagulation function and triglyceride metabolism.

Asunto(s)

Riñón , Ratas Sprague-Dawley , Animales , Masculino , Administración Oral , Riñón/efectos de los fármacos , Riñón/patología , Ratas , Asarum/química , Hígado/efectos de los fármacos , Hígado/patología , Cápsulas , Ácidos Aristolóquicos/toxicidad , Ácidos Aristolóquicos/administración & dosificación , Medicamentos Herbarios Chinos/toxicidad , Medicamentos Herbarios Chinos/administración & dosificación , Femenino , Espectrometría de Masas en Tándem

RESUMEN

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 (HSAMYR) and its AA complexes (HSAMYR/AA-I and HSAMYR/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 · 106 M-1 for HSA/AA-I versus 8.4 and 9.0 · 105 M-1 for HSA/AA-II. Fluorescence quenching of the HSA Trp214 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.

Asunto(s)

Ácidos Aristolóquicos , Albúmina Sérica Humana , Ácidos Aristolóquicos/metabolismo , Ácidos Aristolóquicos/química , Humanos , Cristalografía por Rayos X , Albúmina Sérica Humana/metabolismo , Albúmina Sérica Humana/química , Aductos de ADN/metabolismo , Aductos de ADN/química , Unión Proteica , Ácido Mirístico/metabolismo , Ácido Mirístico/química

RESUMEN

Aristolochic acid (AA)-IIIa is an AA analog present in Aristolochiaceae plants. To evaluate the chronic toxicity of AA-IIIa, mice were intragastrically administered with media control, 1 mg/kg AA-IIIa, and 10 mg/kg AA-IIIa, and designated as the control (CTL), AA-IIIa low dose (AA-IIIa-L), and AA-IIIa high dose (AA-IIIa-H) groups, respectively. AA-IIIa was administered three times a week, every other day, for 24 weeks (24-week time point). Thereafter, some mice were sacrificed immediately, while others were sacrificed 29 or 50 weeks after AA-IIIa withdrawal (53- or 74-week time point). Serum and organs were collected for biochemical and pathological analyses, respectively. Whole-genome sequencing was performed on the kidney, liver, and stomach tissues of AA-IIIa-treated mice for single-nucleotide polymorphism (SNP) detection. AA-IIIa-H mice died at 66 weeks, and the remaining mice showed moribund conditions at the 69 weeks. AA-IIIa induced minor kidney tubule injury, fibroblast hyperplasia, and forestomach carcinoma in mice. Bladder, intestine, liver, heart, spleen, lung, and testis tissues were not pathologically altered by AA-IIIa. In addition, AA-IIIa increased the C:G > A:T mutation in the kidney; however, no SNP mutation changes were observed in the liver and forestomach tissues of AA-IIIa-H mice at the 24-week time point compared with control mice. Therefore, we suspect that AA-IIIa is potentially mutagenic for mice after overdose and long-term administration. On the other hand, the forestomach is a unique organ in mice, but it does not exist in humans; thus, we hypothesize that the stomach toxicity induced by AA-IIIa is not a suitable reference for toxicological evaluation in humans. We recommend that Aristolochiaceae plants containing AA-IIIa should be properly supervised, and overdosing and long-term administration of drugs containing AA-IIIa should be avoided.

Asunto(s)

Ácidos Aristolóquicos , Animales , Ácidos Aristolóquicos/toxicidad , Ratones , Masculino , Riñón/efectos de los fármacos , Riñón/patología , Polimorfismo de Nucleótido Simple , Femenino , Hígado/efectos de los fármacos , Hígado/patología , Estómago/efectos de los fármacos , Estómago/patología , Pruebas de Toxicidad Crónica/métodos , Relación Dosis-Respuesta a Droga

RESUMEN

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

Asunto(s)

Ácidos Aristolóquicos , Colesterol , Ácido Flufenámico , Receptores Acoplados a Proteínas G , Gusto , Humanos , Ácidos Aristolóquicos/metabolismo , Ácidos Aristolóquicos/química , Ácidos Aristolóquicos/farmacología , Sitios de Unión/efectos de los fármacos , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacología , Microscopía por Crioelectrón , Ácido Flufenámico/química , Ácido Flufenámico/metabolismo , Ácido Flufenámico/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestructura , Gusto/efectos de los fármacos , Gusto/fisiología , Transducina/química , Transducina/metabolismo

RESUMEN

BACKGROUND: Aristolochic acid nephropathy (AAN) is a rapidly progressive interstitial nephropathy caused by Aristolochic acid (AA). AAN is associated with the development of nephropathy and urothelial carcinoma. It is estimated that more than 100 million people worldwide are at risk of developing AAN. However, the underlying mechanisms driving renal deterioration in AAN remain poorly understood, and the treatment options are limited. METHODS: We obtained GSE27168 and GSE136276 series matrix data from the Gene Expression Omnibus (GEO) related to AAN. Using the R Studio environment, we applied the limma package and WGCNA package to identify co-differently expressed genes (co-DEGs). By GO/KEGG/GSVA analysis, we revealed common biological pathways. Subsequently, co-DEGs were subjected to the String database to construct a protein-protein interaction (PPI) network. The MCC algorithms implemented in the Cytohubba plugin were employed to identify hub genes. The hub genes were cross-referenced with the transcription factor (TF) database to identify hub TFs. Immune infiltration analysis was performed to identify key immune cell groups by utilizing CIBERSORT. The expressions of AAN-associated hub TFs were verified in vivo and in vitro. Finally, siRNA intervention was performed on the two TFs to verify their regulatory effect in AAN. RESULTS: Our analysis identified 88 co-DEGs through the "limma" and "WGCNA" R packages. A PPI network comprising 53 nodes and 34 edges was constructed with a confidence level >0.4. ATF3 and c-JUN were identified as hub TFs potentially linked to AAN. Additionally, expressions of ATF3 and c-JUN positively correlated with monocytes, basophils, and vessels, and negatively correlated with eosinophils and endothelial cells. We observed a significant increase in protein and mRNA levels of these two hub TFs. Furthermore, it was found that siRNA intervention targeting ATF3, but not c-JUN, alleviated cell damage induced by AA. The knockdown of ATF3 protects against oxidative stress and inflammation in the AAN cell model. CONCLUSION: This study provides novel insights into the role of ATF3 in AAN. The comprehensive analysis sheds light on the molecular mechanisms and identifies potential biomarkers and drug targets for AAN treatment.

Asunto(s)

Ácidos Aristolóquicos , Enfermedades Renales , Factores de Transcripción , Ácidos Aristolóquicos/toxicidad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Enfermedades Renales/inducido químicamente , Enfermedades Renales/genética , Animales , Ratones , Humanos , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Mapas de Interacción de Proteínas