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.

Study on the potential nephrotoxicity and mutagenicity of aristolochic acid IVa and its mechanism.

Previous reports demonstrated that aristolochic acids (AAs) exposure-induced nephrotoxicity, mutations, and tumorigenesis are mainly due to aristolochic acid I (AAI). Notably, the chemical structure of aristolochic acid IVa (AAIVa), which exists at higher levels in many Aristolochiaceae herbs, is extremely similar to AAI. In lack of toxicological data, it is unknown whether AAIVa exposure leads to aristolochic acid nephropathy (AAN), mutations, and tumorigenesis as of AAI. To answer these questions, mice were administered AAIVa by single or repeated long-term gavage, while AAI was used as a positive control. We found that single gavage of 40 mg/kg of AAIVa exhibited no obvious toxicity. Also, there were no tumors or death in mice administrated with 1 and 10 mg/kg of AAIVa for 6 months followed by a 12-month recovery time. There were no noteworthy alterations in gene mutation frequency in the kidney, liver, and stomach between the AAIVa and control mice. Fascinatingly, AA-associated mutational signatures, adenine-to-thymine (A>T) transversions, were absent in AAIVa-treated mice. Nonetheless, 10 mg/kg of AAIVa triggered lymphocytic infiltration and slight fibrous hyperplasia in the kidney at the 6th month; however, these were alleviated at the 12th and 18th months. On the contrary, AAI (positive control) caused severe diffuse fibrosis, tubular atrophy, necrosis, tumors in the forestomach and kidney, and death after the 6th month. It seems that long-term AAIVa exposure induced mild renal lesions could be due to the activation of the canonical or noncanonical transforming growth factor-ß (TGFß) pathway. Overall, these findings suggest that the mutagenicity and carcinogenic risk of AAIVa are very low.

Aristolochic acid IVa forms DNA adducts in vitro but is non-genotoxic in vivo.

Aristolochic acids (AAs) are a family of natural compounds with AA I and AA II being known carcinogens, whose bioactivation causes DNA adducts formation. However, other congeners have rarely been investigated. This study aimed to investigate genotoxicity of AA IVa, which differs from AA I by a hydroxyl group, abundant in Aristolochiaceae plants. AA IVa reacted with 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) to form three dA and five dG adducts as identified by high-resolution mass spectrometry, among which two dA and three dG adducts were detected in reactions of AA IVa with calf thymus DNA (CT DNA). However, no DNA adducts were detected in the kidney, liver, and forestomach of orally dosed mice at 40 mg/kg/day for 2 days, and bone marrow micronucleus assay also yielded negative results. Pharmacokinetic analyses of metabolites in plasma indicated that AA IVa was mainly O-demethylated to produce a metabolite with two hydroxyl groups, probably facilitating its excretion. Meanwhile, no reduced metabolites were detected. The competitive reaction of AA I and AA IVa with CT DNA, with adducts levels varying with pH of reaction revealed that AA IVa was significantly less reactive than AA I, probably by hydroxyl deprotonation of AA IVa, which was explained by theoretical calculations for reaction barriers, energy levels of the molecular orbits, and charges at the reaction sites. In brief, although it could form DNA adducts in vitro, AA IVa was non-genotoxic in vivo, which was attributed to its low reactivity and biotransformation into an easily excreted metabolite rather than bioactivation.

Cytotoxicity and genotoxicity of the carcinogen aristolochic acid I (AA-I) in human bladder RT4 cells.

Aristolochic acid (AA-I) induces upper urothelial tract cancer (UUTC) and bladder cancer (BC) in humans. AA-I forms the 7-(2'-deoxyadenosin-N6-yl)aristolactam I (dA-AL-I) adduct, which induces multiple A:T-to-T:A transversion mutations in TP53 of AA-I exposed UTUC patients. This mutation is rarely reported in TP53 of other transitional cell carcinomas and thus recognized as an AA-I mutational signature. A:T-to-T:A transversion mutations were recently detected in bladder tumors of patients in Asia with known AA-I-exposure, implying that AA-I contributes to BC. Mechanistic studies on AA-I genotoxicity have not been reported in human bladder. In this study, we examined AA-I DNA adduct formation and mechanisms of toxicity in the human RT4 bladder cell line. The biological potencies of AA-I were compared to 4-aminobiphenyl, a recognized human bladder carcinogen, and several structurally related carcinogenic heterocyclic aromatic amines (HAA), which are present in urine of smokers and omnivores. AA-I (0.05-10 µM) induced a concentration- and time-dependent cytotoxicity. AA-I (100 nM) DNA adduct formation occurred at over a thousand higher levels than the principal DNA adducts formed with 4-ABP or HAAs (1 µM). dA-AL-I adduct formation was detected down to a 1 nM concentration. Studies with selective chemical inhibitors provided evidence that NQO1 is the major enzyme involved in AA-I bio-activation in RT4 cells, whereas CYP1A1, another enzyme implicated in AA-I toxicity, had a lesser role in bio-activation or detoxification of AA-I. AA-I DNA damage also induced genotoxic stress leading to p53-dependent apoptosis. These biochemical data support the human mutation data and a role for AA-I in BC.

Quantitation of Protein Adducts of Aristolochic Acid I by Liquid Chromatography-Tandem Mass Spectrometry: A Novel Method for Biomonitoring Aristolochic Acid Exposure.

Emerging evidence suggests that chronic exposure to aristolochic acids (AAs) is one of the etiological pathways leading to chronic kidney disease (CKD). Due to the traditional practice of herbal medicine and AA-containing plants being used extensively as medicinal herbs, over 100 million East Asians are estimated to be at risk of AA poisoning. Given that the chronic nephrotoxicity of AAs only manifests itself after decades of exposure, early diagnosis of AA exposure could allow for timely intervention and disease risk reduction. However, an early detection method is not yet available, and diagnosis can only be established at the end stage of CKD. The goal of this study was to develop a highly sensitive and selective method to quantitate protein adducts of aristolochic acid I (AAI) as a biomarker of AA exposure. The method entails the release of protein-bound aristolactam I (ALI) by heat-assisted alkaline hydrolysis, extraction of ALI, addition of internal standard, and quantitation by liquid chromatography-tandem mass spectrometric analysis. Accuracy and precision of the method were critically evaluated using a synthetic ALI-containing glutathione adduct. The validated method was subsequently used to detect dose-dependent formation of ALI-protein adducts in human serum albumin exposed to AAI and in proteins isolated from the tissues and sera of AAI-exposed rats. Our time-dependent study showed that ALI-protein adducts remained detectable in rats even at 28 days postdosing. It is anticipated that the developed method will fill the technical gap in diagnosing AA intoxication and facilitate the biomonitoring of human exposures to AAs.

In Vivo Metabolism of Aristolochic Acid I and II in Rats Is Influenced by Their Coexposure.

The plant extract aristolochic acid (AA), containing aristolochic acid I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy and Balkan endemic nephropathy, unique renal diseases associated with upper urothelial cancer. Differences in the metabolic activation and detoxification of AAI and AAII and their effects on the metabolism of AAI/AAII mixture in the plant extract might be of great importance for an individual's susceptibility in the development of AA-mediated nephropathies and malignancies. Here, we investigated in vivo metabolism of AAI and AAII after ip administration to Wistar rats as individual compounds and as AAI/AAII mixture using high performance liquid chromatography/electrospray ionization mass spectrometry. Experimental findings were supported by theoretical calculations using density functional theory. We found that exposure to AAI/AAII mixture affected the generation of their oxidative and reductive metabolites formed during Phase I biotransformation and excreted in rat urine. Several Phase II metabolites of AAI and AAII found in the urine of exposed rats were also analyzed. Our results indicate that AAI is more efficiently metabolized in rats in vivo than AAII. Whereas AAI is predominantly oxidized during in vivo metabolism, its reduction is the minor metabolic pathway. In contrast, AAII is mainly metabolized by reduction. The oxidative reaction only occurs if aristolactam II, the major reductive metabolite of AAII, is enzymatically hydroxylated, forming aristolactam Ia. In AAI/AAII mixture, the metabolism of AAI and AAII is influenced by the presence of both AAs. For instance, the reductive metabolism of AAI is increased in the presence of AAII while the presence of AAI decreased the reductive metabolism of AAII. These results suggest that increased bioactivation of AAI in the presence of AAII also leads to increased AAI genotoxicity, which may critically impact AAI-mediated carcinogenesis. Future studies are needed to explain the underlying mechanism(s) for this phenomenon.

Recognition of the toxicity of aristolochic acid.

WHAT IS KNOWN AND OBJECTIVE: Aristolochic acid (AA) is an abundant compound in Aristolochia plants and various natural herbs. In the 1990s, a slimming formula used in Belgium that contains Aristolochia fangchi was reported to cause kidney damage and bladder cancer, and aristolochic acid nephropathy (AAN) is now well recognized worldwide. In October 2017, researchers reported an AA signature that is closely associated with hepatocellular carcinoma (HCC) worldwide. COMMENT: There are differing opinions on the toxicity of AA, and different countries have taken different measures to address the issue. There is a lack of clarity on the causal role of AA in hepatocarcinogenesis and on the potential underlying mechanisms for the reported nephrotoxicity and carcinogenicity. The toxicity of AA differs depending on gender and age, and other risk factors that could explain the variability in the toxicity of AA remain to be identified. WHAT IS NEW AND CONCLUSION: Whether preparations containing AA, such as many Chinese medicines, should be used remains controversial, and this issue warrants further investigation before definite conclusions can be drawn.

Urinary Time- or Dose-Dependent Metabolic Biomarkers of Aristolochic Acid-Induced Nephrotoxicity in Rats.

The metabolic mechanisms underlying aristolochic acid (AA)-induced nephrotoxicity are inconclusive. A Gas Chromatography-Mass Spectrometer (GC-MS)-based metabolomic study was performed to analyze urinary metabolites in AA-treated rats at different dosages (10, 20, and 40 mg/kg) and time points (2, 4, and 6 days). Serum blood urea nitrogen (BUN), creatinine, and kidney injury were significantly changed only on the 6th day in 40 mg/kg AA group, whereas metabolic alternation appeared even on the 2nd day in 10 mg/kg AA group. A total of 84 differential metabolites were identified in 40 mg/kg AA groups time-dependently and 81 in 10, 20, and 40 mg/kg AA groups dose-dependently (6 days) compared with control group. Eight metabolites were selected as potential metabolic biomarkers including methylsuccinic acid, nicotinamide, 3-hydroxyphenylacetic acid, citric acid, creatinine, uric acid, glycolic acid, and gluconic acid. Four of them were dose-dependently altered including methylsuccinic acid, citric acid, creatinine, and 3-hydroxyphenylacetic acid, which were defined as "early metabolic biomarker." The alteration of nicotinamide, uric acid, and gluconic acid was time- and dose-dependent, whereas the change of glycolic acid was time- or dose-independent. The latter 4 metabolites were defined as "late metabolic biomarker" because of the obvious reduction on the 6th day in 40 mg/kg AA group. In summary, the urinary metabolic alterations were more sensitive than conventional biomarkers of renal injury. The identified metabolites suggested pathways of energy metabolism, gut microbiota, and purine metabolism were associated with AA-induced nephrotoxicity time- or dose-dependently. Further investigation was warranted to determine the roles of the 8 potential metabolic biomarkers in AA-induced nephrotoxicity.

Traditional Chinese medicine: Pivotal role of the spleen in the metabolism of aristolochic acid I in rats is dependent on oatp2a1.

The genotoxicity and cytotoxicity of aristolochic acids is well documented, and the Aristolochiaceae plant family has been widely used in China and India for medical purposes. However, the mechanisms of aristolochic acid I (AAI) in treatment and toxicity remain to be fully elucidated. According to the theory of traditional Chinese medicine (TCM), the spleen is responsible for transportation and transformation, in which a substance is transformed, absorbed and distributed in the body. In the present study, rats were randomized into a blank group without spleen deficiency and a spleen deficiency group to investigate the metabolism of AAI. The results showed that the concentration of AAI was higher in the spleen deficiency group, compared with that of the blank group. To further elucidate this process, the expression of organic anion transporting peptide (oatp)2a1 in the rats of the two groups were examined following oral administration of AAI. It was observed that the mRNA level of oatp2a1 in the small intestine of the blank+AAI 60 min group was downregulated, compared with that in the blank group. Compared with the mRNA level of oatp2a1 in the spleen deficiency group, the expression levels in the lung and liver were downregulated in the spleen deficiency+AAI 5 min group, whereas expression levels in the kidney in the spleen deficiency+AAI 60 min group were upregulated. Based on the above results, it was hypothesized that the expression of oatp2a1 may be one of the mechanisms of AAI metabolism in rats. In TCM, the spleen and certain functions of the small intestine, are important in AAI metabolism, and affect the toxicity of AAI. In addition, the lung, liver and kidney may also be involved in spleen deficiency syndrome in rats.

Hepatic Premalignant Alterations Triggered by Human Nephrotoxin Aristolochic Acid I in Canines.

Aristolochic acid I (AAI) existing in plant drugs from Aristolochia species is an environmental human carcinogen associated with urothelial cancer. Although gene association network analysis demonstrated gene expression profile changes in the liver of human TP53 knock-in mice after acute AAI exposure, to date, whether AAI causes hepatic tumorigenesis is still not confirmed. Here, we show that hepatic premalignant alterations appeared in canines after a 10-day AAI oral administration (3 mg/kg/day). We observed c-Myc oncoprotein and oncofetal RNA-binding protein Lin28B overexpressions accompanied by cancer progenitor-like cell formation in the liver by AAI exposure. Meanwhile, we found that forkhead box O1 (FOXO1) was robustly phosphorylated, thereby shuttling into the cytoplasm of hepatocytes. Furthermore, utilizing microarray and qRT-PCR analysis, we confirmed that microRNA expression significantly dysregulated in the liver treated with AAI. Among them, we particularly focused on the members in let-7 miRNAs and miR-23a clusters, the downstream of c-Myc and IL6 receptor (IL6R) signaling pathway linking the premalignant alteration. Strikingly, when IL6 was added in vitro, IL6R/NF-κB signaling activation contributed to the increase of FOXO1 phosphorylation by the let-7b inhibitor. Therefore, it highlights the new insight into the interplay of the network in hepatic tumorigenesis by AAI exposure, and also suggests that anti-premalignant therapy may be crucial for preventing AAI-induced hepatocarcinogenesis.

Tissue-specific microRNA responses in rats treated with mutagenic and carcinogenic doses of aristolochic acid.

Aristolochic acid (AA) is an active component in herbal drugs derived from the Aristolochia species. Although these drugs have been used since antiquity, AA is both genotoxic and carcinogenic in animals and humans, resulting in kidney tumours in rats and upper urinary tract tumours in humans. In the present study, we conducted microarray analysis of microRNA (miRNA) expression in tissues from transgenic Big Blue rats that were treated for 12 weeks with 0.1-10mg/kg AA, using a protocol that previous studies indicate eventually results in kidney tumours and mutations in kidney and liver. Global analysis of miRNA expression of rats treated with 10 mg/kg AA indicated that 19 miRNAs were significantly dysregulated in the kidney, with most of the miRNAs related to carcinogenesis. Only one miRNA, miR-34a (a tumour suppressor), was differentially expressed in the liver. The expression of the two most responsive kidney miRNAs (miR-21, an oncomiR and miR-34a) was further examined in the kidney, liver and testis of rats exposed to 0, 0.1, 1.0 and 10mg/kg AA. Expression of miR-21 was up-regulated in the kidney only, while miR-34a was dose-dependently up-regulated in both the kidney and liver; the expression of miR-21 and miR-34a was unaltered by the AA treatment in the testis. Analysis of cII mutations in the testis of treated rats also was negative. Our results indicate that AA treatment of rats produced dysregulation of a large number of miRNAs in the tumour target tissue and that the up-regulation of miR-21 correlated with the carcinogenicity of AA while the up-regulation of miR-34a correlated with its mutagenicity.

Variation in presentation and presence of DNA adducts and p53 mutations in patients with endemic nephropathy--an environmental form of the aristolochic acid nephropathy.

BACKGROUND: Endemic nephropathy (EN) and associated urothelial cell cancers (UUC) are an environmental form of aristolochic acid nephropathy where the most probable rout of ingestion of aristolochic acid (AA) was made by bread contaminated with AA, leading to chronic dietary intoxication. Clinical courses of three members of the same family, similarly exposed to toxin, who exhibited different clinical courses of the disease are presented. METHODS: Questionnaires on AA exposure were taken. Tissue samples were obtained during therapeutic nephrouretectomies. Histopathology, immunohistochemical detection of p53, p53 mutation screening in tumor DNA and analysis on the presence of aristolactam (AL)-DNA adducts were performed. RESULTS: Case 1 had UUC with typical EN histopathological signs, whereas Case 2 had bilateral UUCs with typical EN histopathological signs. In contrast, the patient in Case 3 initially showed renal insufficiency, complicated afterwards by right UUC, and later on by left UUC with histopathological end-stage chronic changes but without typical EN changes. AA-DNA adducts and specific p53 mutational spectra (A:T→ T:A transversion) were found in tissues of cases 1 and 2. CONCLUSION: Diverse clinical courses seem to be related not to differences in exposure but to differences in metabolic activation or detoxification of AA and/or DNA repair resulting from different genetic polymorphisms.

Risk assessment of upper tract urothelial carcinoma related to aristolochic acid.

BACKGROUND: Aristolochic acid is a toxin found in plants of the genus Aristolochia, to which humans can be exposed either through certain Chinese herbal medicines or through inadvertent commingling with food crops. Our objective was to estimate cumulative exposures of aristolochic acid associated with increased risk of end-stage renal disease (ESRD), and to conduct a systematic review and meta-analysis on aristolochic acid-induced upper tract urothelial carcinoma (UUC). METHODS: Using epidemiologic studies on aristolochic acid-related disease from multiple different regions of the world, a systematic review was conducted in which relative risks (RR), HRs, and ORs were derived or extracted directly, and a meta-analysis was conducted. One study was used to estimate a benchmark dose lower confidence limit (BMDL) for aristolochic acid-related ESRD. RESULTS: Mean values for risk ratios, ORs, RRs, or HRs, of UUC caused by aristolochic acid ranged from 1 to 49. A meta-analysis of these studies resulted in a pooled OR of 5.97 [95% confidence interval (CI), 2.78-12.84] for this aristolochic acid-related cancer. The obtained BMDL for aristolochic acid-related ESRD was 0.42 g cumulative aristolochic acid exposure. CONCLUSIONS: Aristolochic acid exposure is significantly associated with an increased risk of UUC, and there is a dose-dependent relationship between cumulative aristolochic acid exposure and ESRD risk. IMPACT: Individuals who use certain Chinese herbal medicines may significantly increase their risk of developing UUC and/or ESRD, as would individuals who are inadvertently exposed to aristolochic acid through commingling of Aristolochia plants with harvested food crops.

[Development of gastric precancerous lesion animal model].

OBJECTIVE: To establish a model of gastric precancerous lesion by using Aristolochic manshuriensis which contains aristolochic acids. METHOD: The SD rats were randomly divided into four groups: control and three different doses of ethanol extractive of A. manshuriensis (EEA) (corresponding to aristolochic acid I 2.5, 5.0, 10.0 mg x kg(-1)), respectively. EEA was intragastrically given to rats every other day. At the end of the 10th, 15th, 20th week, part of the rats in each group was sacrificed and the stomachs were weighed. The gastric tumor was assessed by the weight and the relative stomach weight to the body weight. The stomachs were fixed in 4% neutral formalin, and the paraffin imbedding tissues were sliced and HE stained. Histomorphology was observed under the light microscope to determine gastric hyperplasia, mucosa precancerosis (atypical hyperplasia) and gastric cancer formation. RESULT: The rats treated with different doses of EEA for 10 weeks induced mucosa papillary, epithelioma hyperplasia. Histological observation showed mucosa precancerosis lesions characterized as atypical hyperplasia at the dose levels corresponding to aristolochic acid I 5.0 and 10.0 mg x kg(-1) treated for 10 weeks. The incidence rate of gastric precancerosis in those two groups was 100% at the 15th week. Malignant tumors were observed in most of the animals in 10.0 mg x kg(-1) group. The animals in 5.0 mg x kg(-1) group were well tolerant compared to 10.0 mg x kg(-1) group during the course of experiment, so the dose of aristolochic acid I 5.0 mg x kg(-1) and 10-15 weeks treatment were considered to be optimum to establish the model of gastric precancerosis. CONCLUSION: A rat model of gastric precancerosis can be induced within a short duration by giving an oral administration of the ethanol extract of A. manshuriensis which contains aristolochic acids.

Developmental nephrotoxicity of aristolochic acid in a zebrafish model.

Aristolochic acid (AA) is a component of Aristolochia plant extracts which is used as a treatment for different pathologies and their toxicological effects have not been sufficiently studied. The aim of this study was to evaluate AA-induced nephrotoxicity in zebrafish embryos. After soaking zebrafish embryos in AA, the embryos displayed malformed kidney phenotypes, such as curved, cystic pronephric tubes, pronephric ducts, and cases of atrophic glomeruli. The percentages of embryos with malformed kidney phenotypes increased as the exposure dosages of AA increased. Furthermore, AA-treated embryos exhibited significantly reduced glomerular filtration rates (GFRs) in comparison with mock-control littermates (mock-control: 100±2.24% vs. 10 ppm AA treatment for 3-5h: 71.48±18.84%~39.41±15.88%), indicating that AA treatment not only caused morphological kidney changes but also induced renal failure. In addition to kidney malformations, AA-treated zebrafish embryos also exhibited deformed hearts, swollen pericardiums, impaired blood circulation and the accumulation(s) of red blood cells. Whole-mount in situ hybridization studies using cmlc2 and wt1b as riboprobes indicated that the kidney is more sensitive than the heart to AA damage. Real-time PCR showed that AA can up-regulate the expression of proinflammatory genes like TNFα, cox2 and mpo. These results support the following conclusions: (1) AA-induced renal failure is mediated by inflammation, which causes circulation dysfunction followed by serious heart malformation; and (2) the kidney is more sensitive than the heart to AA injury.

Mutagenicity and DNA adduct formation by aristolochic acid in the spleen of Big Blue® rats.

Aristolochic acid (AA) is a potent human nephrotoxin and carcinogen. We previously reported that AA treatment resulted in DNA damage and mutation in the kidney and liver of rats. In this study, we have determined the DNA adducts and mutations induced by AA in rat spleen. Big Blue® transgenic rats were gavaged with 0, 0.1, 1.0, and 10.0 mg AA/kg body weight five-times/week for 3 months. Three DNA adducts, [7-(deoxyadenosin-N(6)-yl)-aristolactam I, 7-(deoxyadenosin-N(6)-yl)-aristolactam II and 7-(deoxyguanosin-N(2)-yl)-aristolactam I], were identified by (32)P-postlabeling. Over the dose range studied, there were strong linear dose-responses for AA-DNA adduct formation in the treated rat spleens, ranging from 4.6 to 217.6 adducts/10(8) nucleotides. Spleen cII mutant frequencies also increased in a dose-dependent manner, ranging from 32.7 to 286.2 × 10(-6) in the treated animals. Mutants isolated from the different treatment groups were sequenced; analysis of the resulting spectra indicated that there was a significant difference between the pattern of mutation in the 10 mg/kg AA-treated and the vehicle control rats. A:T → T:A transversion was the major type of mutation in AA-treated rats, whereas G:C → A:T transition was the main type of mutation in the vehicle controls. These results indicate that AA is genotoxic in the spleen of rats exposed under conditions that result in DNA adduct formation and mutation induction in kidney and liver.

Interstitial fibrosis is associated with increased COL1A2 transcription in AA-injured renal tubular epithelial cells in vivo.

Accumulation of type I collagen is a key event in renal interstitial fibrosis. As there is no effective treatment, understanding the site where collagen is transcribed and the factors driving it in response to disease in vivo is critical for designing future therapies. The present research investigated the transcriptional activity of the COL1A2 gene in a mouse model of progressive fibrosis induced by aristolochic acid (aristolochic acid nephropathy, AAN). To achieve this we genetically modified mice to express a reporter gene (LacZ) and CCN2 (connective tissue growth factor) under the transcriptional control of the COL1A2 promoter /enhancer sequences. Using these mice we asked where is collagen actively transcribed and secondly, what is the role of CCN2 in AAN. Here, we report that de-novo transcription of the COL1A2 gene occurred predominantly in damaged tubular epithelial cells during progressive interstitial fibrosis in vivo. The activation of COL1A2 was studied by detection of the reporter gene LacZ and COL1A2 mRNA in interstitial, glomerular, vascular, and tubular epithelial tissue from laser capture microscopy. We also demonstrated that LacZ-positive cells co-express E-Cadherin a marker of epithelial origin which is consistent with an epithelial phenotype which is capable of collagen expression during injury. There was no evidence of detachment of these cells from tubules to become myofibroblasts. Moreover, we showed that the transgenic mice show a modest enhancement of CCN2 expression; however fibrosis induced by AA is the same in transgenics and controls suggesting that CCN2, at this level of expression, is not sufficient to enhance fibrogenesis. Overall our study provides a better understanding into the expression patterns and roles of two major extracellular matrix proteins: type I collagen and CCN2.

TP53 Mutational signature for aristolochic acid: an environmental carcinogen.

This study was designed to establish the TP53 mutational spectrum of aristolochic acid (AA), examined in the context of endemic (Balkan) nephropathy, an environmental disease associated with transitional cell (urothelial) carcinomas of the upper urinary tract (UUC). Tumor tissue was obtained from residents of regions in Bosnia, Croatia and Serbia where endemic nephropathy has been prevalent for over 50 years. Fifty-nine TP53 mutations were detected in 42 of the 97 tumors analyzed. Mutational spectra were dominated by A:T to T:A transversions with the mutated adenines located almost exclusively on the nontranscribed strand. This marked strand bias is attributed to selective processing of aristolactam-dA adducts by transcription-coupled nucleotide excision repair. Hotspots for A:T to T:A mutations include codons 131 and 179 and the 5'-AG acceptor splice site of intron 6. The unique TP53 mutational signature for AA identified in this study can be used to explore the hypothesis that botanical products containing this human carcinogen and nephrotoxin are responsible, in part, for the high prevalence of UUC and chronic renal disease in countries where Aristolochia herbal remedies traditionally have been used for medicinal purposes.

Aristolochic acid I induced oxidative DNA damage associated with glutathione depletion and ERK1/2 activation in human cells.

Aristolochic acid I (AAI) has been widely found in herbal remedies and linked to the development of nephropathy and urothelial carcinoma in humans. This study elucidated the mechanism of oxidative stress and DNA damage mediated by AAI in human cells. Treatment of human promyelocytic leukemia cells (HL-60) and human renal proximal tubular cells (HK-2) with AAI led to a dose-dependent increase of reactive oxygen species (ROS). AAI also elevated the levels of DNA strand breaks and 8-hydroxy guanosine in HL-60 and HK-2 cells. Antioxidants, including Tiron, N-acetyl-l-cysteine (NAC) and glutathione (GSH), effectively suppressed the AAI-induced ROS and AAI-elicited genotoxicity, indicating that AAI induced the DNA damage through oxidative stress. GSH depletion was also found in AAI-treated cultures and proceeded prior to ROS formation. Exposure of HL-60 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only MEK1/2 inhibitor, U0126, significantly decreased AAI-mediated ROS. Preincubation of cells with thiol-containing compounds (NAC and GSH) inhibited the caspase 3 activity triggered by AAI, but non-thiol Tiron did not show a similar effect. This study demonstrated that AAI treatment results in oxidative stress-related DNA damage through GSH depletion and ERK1/2 activation; AAI-induced apoptosis is associated with GSH loss, but is independent of ROS generation.

Therapeutic remedies based on Aristolochia clematitis in the main foci of Balkan endemic nephropathy in Romania.

Balkan endemic nephropathy (BEN) is a disease found in Romania and neighboring countries in the Balkan area. In Romania, BEN is most prevalent in Mehedinti County, located in the South of Romania near the Danube River. The etiology of the disease is as yet unknown. One of the current hypotheses concerning BEN etiology is an involvement of aristolochic acid (AA). BEN bears many similarities to aristolochic nephropathy, which is developed due to the use of Chinese herbs as therapeutic remedies in slimming diets. This paper analyzes the involvement of therapeutic remedies based on AA in the BEN found in Mehedinti County, where these herbs have been traditionally used. The presence of AA in the plasma of BEN patients as well as of other subjects, including healthy relatives of these patients and other persons from the BEN-affected area, has been analyzed. No AA was detected in the plasma of the studied subjects. This proves the absence, at the current time, of an AA contribution in the analyzed subjects. Therapeutic remedies based on AA have been used in the BEN-affected area. We were not able to reveal direct relationships between these remedies and either the development of BEN in dialyzed patients or the development of urinary-tract tumors in dialyzed patients with urothelial tumors. Therapeutic remedies based on Aristolochiaclematitis may play a stimulating role in BEN with regard to its development and the development of urinary-tract tumors. There may be a relationship between BEN and cumulative previous exposure to low doses of AA due to the consumption of contaminated foodstuffs, which could add to any contributions by therapeutic remedies.