Positive Feedback Mechanism in Aristolochic Acid I Exposure-Induced Anemia and DNA Adduct Formation: Implications for Balkan Endemic Nephropathy.

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.

Long-term toxicity evaluation of aristolochic acid-IIIa in mice.

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.

Identification and analysis of differently expressed transcription factors in aristolochic acid nephropathy.

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.

Serum Amyloid A3 Promoter-Luciferase Reporter Mice Are Useful for Early Drug-Induced Nephrotoxicity Detection.

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.

Long-term oral administration of Kelisha capsule does not cause hepatorenal toxicity in rats.

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.

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.

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

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic 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 STUDY: To 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 METHODS: The 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. RESULTS: AAD 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-ß-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. CONCLUSION: Collectively, 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.

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 nonbarrier 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 2 mg/kg aristolochic acid (AA) injections intraperitoneally to induce CKD or saline injections as a control. We evaluated their kidney immune profiles at 2 wk, 6 wk, and 6 mo 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 (sc) RNA sequencing revealed these cells as expressing well-known TRM transcription factors and receptors responsible for TRM differentiation and maintenance. Although 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 nonantigen-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.NEW & NOTEWORTHY Resident memory T cells (TRMs) differentiate and are retained within the kidneys of mice with aristolochic acid (AA)-induced chronic kidney disease (CKD). Here, we characterized this kidney TRM population and demonstrated TRM derivation in the kidneys of OT-1 transgenic mice with AA-induced CKD. A better understanding of TRMs and the processes by which they can differentiate independent of antigen may help our understanding of the interactions between the immune system and kidneys.

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.

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.

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.

SGLT2 inhibitor dapagliflozin protects the kidney in a murine model of Balkan nephropathy.

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.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSION AND IMPLICATIONS: 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.

Aristolochic acid I aggravates oxidative stress-mediated apoptosis by inhibiting APE1/Nrf2/HO-1 signaling.

Nephrotoxicity induced by aristolochic acid I (AAI) is related to redox stress and apoptosis. Apurinic/apyrimidine endonuclease 1 (APE1) has antioxidant and anti-apoptotic effects. This study investigated the potential role of APE1 in AAI-induced nephrotoxicity. Renal injury was successfully induced in C57BL/6J mice by intraperitoneal injection of AAI every other day for 28 days. Expressions of APE1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) in renal tissues of the model mice was inhibited, accompanied by oxidative damage and apoptosis. Similar results were obtained in vitro in human proximal tubular (HK-2) cells damaged by AAI. In the presence of a low concentration of the APE1 inhibitor E3330, expression of Nrf2 and HO-1 proteins in HK-2 cells was decreased and AAI-induced apoptosis was aggravated. Overexpression of APE1 in HK-2 cells promoted the expression of Nrf2 and HO-1, and alleviated apoptosis and renal injury induced by AAI. The collective findings demonstrate that AAI can inhibit the induction of oxidative stress and apoptosis by the APE1/Nrf2/HO-1 axis, leading to AAI renal injury. Targeting APE1 may be an effective therapeutic strategy to treat AA nephrotoxicity.

Investigation of the applicability of the zebrafish model for the evaluation of aristolochic acid-related nephrotoxicity.

BACKGROUND: The risk of compounds/drugs, including aristolochic acid-induced nephrotoxicity remains high and is a significant public health concern. Therefore, it is particularly important to select reasonable animal models for rapid screening and evaluation of different samples with complex chemical systems. The zebrafish (Danio rerio) has been used to study chemical-induced renal toxicity. However, most of the published literature was performed on individual components or drugs, and the key evidence confirming the applicability of zebrafish larvae for the evaluation of aristolochic acid-related nephrotoxicity in complex chemical systems, such as in traditional Chinese medicine (TCM), was insufficient. METHODS: High-performance liquid chromatography (HPLC) was used to determine the content of aristolochic acid (AA) in herbs and Chinese patent medicines. The zebrafish larvae at 4 days post-fertilization (dpf) were used to evaluate the nephrotoxicity of various samples, respectively, based on the phenotype of the kidney and histological, and biochemical. Transcriptome technology was used to investigate the related signaling pathways and potential mechanisms after treatment with AA, which was verified by RT-PCR technology. RESULTS: The results showed that the total amounts of AAI, AAII, and ALI ranged from 0.0004 to 0.1858 g·g-1( %) from different samples, including Aristolochia debilis, Fibraurea recisa, Asarum, Wantongjingu tablets, Jiuweiqianghuo granules, and Xiaoqinglong granules in descending order. Moreover, compared with the negative/blank control, substantial changes in phenotype, histomorphology and biochemical parameters of renal function were observed in the groups challenged with the sublethal concentration of drugs. The transcriptomics results showed the upregulation of most genes in PERK/ATF4/CHOP, ATM/Chk2/p53, Caspase/Bax/Bcl-2a, TGF/Smad/ERK, PI3K/Akt, induced by aristolochic acid analogues, which were essentially consistent with those of the q-RT-PCR experiments, highlighting the similar toxicity response to the previously published article with the other traditional evaluation model. CONCLUSION: The stability, accuracy and feasibility of the zebrafish larval model in screening and evaluating the nephrotoxicity of TCM were validated for the first time on the AAs-related drugs in a unified manner, confirming and promoting the applicability of zebrafish in assessing nephrotoxicity of samples with complex chemical character.

MiR-766-3p and miR-671-5p attenuate aristolochic acid-induced hepatotoxicity by directly targeting the key bioactivating enzyme NQO1.

Aristolochic acid (AA) as an emerging contaminant in herbal medicines or crops has been well-recognized for causing nephropathy since 1990s. Over the last decade, mounting evidence has linked AA to liver injury; however, the underlying mechanism is poorly elucidated. MicroRNAs respond to environmental stress and mediate multiple biological processes, thus showing biomarker potentials prognostically or diagnostically. In the present study, we investigated the role of miRNAs in AA-induced hepatotoxicity, specifically in regulating NQO1, the key enzyme responsible for AA bioactivation. In silico analysis showed that hsa-miR-766-3p and hsa-miR-671-5p were significantly associated with AAI exposure as well as NQO1 induction. A 28-day rat experiment of 20 mg/kg AA exposure demonstrated a 3-fold increase of NQO1 and an almost 50 % decrease of the homologous miR-671 that were accompanied with liver injury, which was consistent with in silico prediction. Further mechanistic investigation using Huh7 cells with IC50 of AAI at 146.5 µM showed both hsa-miR-766-3p and hsa-miR-671-5p were able to directly bind to and down-regulate NQO1 basal expression. In addition, both miRNAs were shown to suppress AAI-induced NQO1 upregulation in Huh7 cells at a cytotoxic concentration of 70 µM, and consequently alleviate AAI-induced cellular effects, including cytotoxicity and oxidative stress. Together, these data illustrate that miR-766-3p and miR-671-5p attenuate AAI-induced hepatotoxicity, and thus have monitoring and diagnostic potentials.

Simultaneous toxicokinetic studies of aristolochic acid I and II and aristolactam I and II using a newly-developed microdialysis liquid chromatography-tandem mass spectrometry.

Aristolochic acids (AAs) are naturally occurring genotoxic carcinogens linked to Balkan endemic nephropathy and aristolochic acid nephropathy. Aristolochic acid I and II (AA-I and AA-II) are the most abundant AAs, and AA-I has been reported to be more genotoxic and nephrotoxic than AA-II. This study aimed to explore metabolic differences underlying the differential toxicity. We developed a novel microdialysis sampling coupled with solid-phase extraction liquid chromatography-tandem mass spectrometry (MD-SPE-LC-MS/MS) to simultaneously study the toxicokinetics (TK) of AA-I and AA-II and their corresponding aristolactams (AL-I and AL-II) in the blood of Sprague Dawley rats co-treated with AA-1 and AA-II. Near real-time monitoring of these analytes in the blood of treated rats revealed that AA-I was absorbed, distributed, and eliminated more rapidly than AA-II. Moreover, the metabolism efficiency of AA-I to AL-I was higher compared to AA-II to AL-II. Only 0.58% of AA-I and 0.084% of AA-II was reduced to AL-I and AL-II, respectively. The findings are consistent with previous studies and support the contention that differences in the in vivo metabolism of AA-I and AA-II may be critical factors for their differential toxicities.

An unprecedented free radical mechanism for the formation of DNA adducts by the carcinogenic N-sulfonated metabolite of aristolochic acids.

The carcinogenicity of aristolochic acids (AAs) has been attributed mainly to the formation of stable DNA-aristolactam (DNA-AL) adducts by its reactive N-sulfonated metabolite N-sulfonatooxyaristolactam (N-OSO3--AL). The most accepted mechanism for such DNA-AL adduct formation is via the postulated but never unequivocally-confirmed aristolactam nitrenium ion. Here we found that both sulfate radical and two ALI-derived radicals (N-centered and C-centered spin isomers) were produced by N-OSO3--ALI, which were detected and unequivocally identified by complementary applications of ESR spin-trapping, HPLC-MS coupled with deuterium-exchange methods. Both the formation of the three radical species and DNA-ALI adducts can be significantly inhibited (up to 90%) by several well-known antioxidants, typical radical scavengers, and spin-trapping agents. Taken together, we propose that N-OSO3--ALI decomposes mainly via a new N-O bond homolysis rather than the previously proposed heterolysis pathway, yielding reactive sulfate and ALI-derived radicals, which are together and in concert responsible for forming DNA-ALI adducts. This study presents strong and direct evidence for the production of free radical intermediates during N-OSO3--ALI decomposition, providing an unprecedented free radical perspective and conceptual breakthrough, which can better explain and understand the molecular mechanism for the formation of DNA-AA adducts, the carcinogenicity of AAs and their potential prevention.

Aristolochic acid I exposure triggers ovarian dysfunction by activating NLRP3 inflammasome and affecting mitochondrial homeostasis.

Aristolochic acids are widely distributed in the plants of Aristolochiaceae family and Asarum species. Aristolochic acid I (AAI) is the most frequent compound of aristolochic acids, which can accumulate in the soil, and then contaminates crops and water and enters the human body. Research has shown that AAI affects the reproductive system. However, the mechanism of AAI's effects on the ovaries at the tissue level still needs to be clarified. In this research, we found AAI exposure reduced the body and ovarian growth in mice, decreased the ovarian coefficient, prevented follicular development, and increased atretic follicles. Further experiments showed that AAI upregulated nuclear factor-κB and tumor necrosis factor-α expression, activated the NOD-like receptor protein 3 inflammasome, and led to ovarian inflammation and fibrosis. AAI also affected mitochondrial complex function and the balance between mitochondrial fusion and division. Metabolomic results also showed ovarian inflammation and mitochondrial dysfunction due to AAI exposure. These disruptions reduced the oocyte developmental potential by forming abnormal microtubule organizing centers and expressing abnormal BubR1 to destroy spindle assembly. In summary, AAI exposure triggers ovarian inflammation and fibrosis, affecting the oocyte developmental potential.

Study on the difference and correlation between the contents and toxicity of aristolochic acid analogues in Aristolochia plants.

ETHNOPHARMACOLOGICAL RELEVANCE: The nephrotoxicity and carcinogenicity induced by traditional Chinese medicines (TCMs) containing aristolochic acids (AAs) and related compound preparations have greatly limited their clinical application. While the toxicity of AA-I and AA-II is relatively clear, there are marked differences in the toxic effects of different types of aristolochic acid analogues (AAAs). Thus, the toxicity of TCMs containing AAAs cannot be evaluated based on the toxicity of a single compound. AIM OF THE STUDY: To systematically investigate the toxicity induced by Zhushalian (ZSL), Madouling (MDL) and Tianxianteng (TXT) as representative TCMs derived from Aristolochia. MATERIALS AND METHODS: AAA contents in ZSL, MDL and TXT were determined using HPLC. Subsequently, mice were treated for 2 weeks with high (H) and low (L) dosages of TCMs containing total AAA contents of 3 mg/kg and 1.5 mg/kg, respectively. Toxicity was evaluated using biochemical and pathological examination and was based on organ indices. Correlations between AAA contents and induced toxicity were analysed using multiple methods. RESULTS: Of the total AAA content, ZSL contained mainly AA-I and AA-II (>90%, of which AA-I accounted for 49.55%). AA-I accounted for 35.45% in MDL. TXT mainly contained AA-IVa (76.84%) and other AAAs accounted for <10%. Short-term toxicity tests indicated that ZSL and high-dose MDL induced obvious renal interstitial fibrosis and gastric injury, whereas TXT (high and low dosages) caused only slight toxicity. Correlation analysis suggested that AA-I might be the critical hazard factor for toxicity. CONCLUSIONS: The toxicity of TCMs containing AAAs cannot be generalised. The toxicity of TXT is relatively low compared with those of ZSL and MDL. The toxicity of Aristolochia depends mainly on the AA-I content; therefore, control of AA-I levels in TCMs and related compound preparations is required to reduce the risk of toxicity associated with the use of Aristolochia herbs in clinical settings.