Biotransformation and Toxicities of Aristolochic Acids.

Environmental and iatrogenic exposures contribute significantly to human diseases, including cancer. The list of known human carcinogens has recently been extended by the addition of aristolochic acids (AAs). AAs occur primarily in Aristolochia herbs, which are used extensively in folk medicines, including Traditional Chinese Medicine. Ingestion of AAs results in chronic renal disease and cancer. Despite importation bans imposed by certain countries, herbal remedies containing AAs are readily available for purchase through the internet. With recent advancements in mass spectrometry, next generation sequencing, and the development of integrated organs-on-chips, our knowledge of cancers associated with AA exposure, and of the mechanisms involved in AA toxicities, has significantly improved. DNA adduction plays a central role in AA-induced cancers; however, significant gaps remain in our knowledge as to how cellular enzymes promote activation of AAs and how the reactive species selectively bind to DNA and kidney proteins. In this review, I describe pathways for AAs biotransformation, adduction, and mutagenesis, emphasizing novel methods and ideas contributing to our present understanding of AA toxicities in humans.

Acute nephrotoxicity of aristolochic acid in vitro: metabolomics study for intracellular metabolic time-course changes.

Time-course metabolic changes of aristolochic acid nephrotoxicity (AAN) was investigated using acute AAN HK-2 model. And the AAN-related biomarkers were selected. In the results, 11 potential identified biomarkers were selected and validated using multivariate method combined with time-course analysis. Several metabolic pathways, including vitamin metabolism, lipids acalytion, trytophan metabolism and protein degradation were found to be associated with AAN pathology. This research will provide a valuable reference for the discovery of more potential biomarkers of AAN progression in clinic.

Anti-nociceptive, anti-inflammatory and toxicological evaluation of Fang-Ji-Huang-Qi-Tang in rodents.

BACKGROUND: Fang-Ji-Huang-Qi-Tang (abbreviated as FJHQT), composed by six medicinal herbs including Radix Stephania Tetrandra, Radix Astragali, Rhizoma Atractylodis Macrocephalae, Radix Glycyrrhizae, Rhizoma Zingiberis and Fructus Ziziphi Jujubae, is a frequently Chinese prescription for treating painful and inflammatory disorders such as rheumatoid arthritis. When Radix Stephania Tetrandra was misused with Aristolochia species, acute or chronic nephropathy caused by aristolochic acid was happened. Thus, the present study was aimed to identify Radix Stephania Tetrandra and performed the pharmacological and toxicological evaluation of FJHQT extract in rodents. METHODS: Radix Stephania Tetrandra was identified by macroscopic and microscopic observation, and the content of tetrandrine in FJHQT extract was measured by high performance liquid chromatography. Then, the pharmacological activities of FJHQT extract with respect to clinical use was investigated with acetic acid-induced writhing response, formalin-induced licking response and carrageenan-induced paw edema. Finally, we evaluated the subacute toxicology of FJHQT extract after 28-day repeated oral administration in rats. RESULTS: Radix Stephania Tetrandra was correctly used in FJHQT extract, and the content of tetrandrine in FJHQT extract was 2.5 mg/g. FJHQT extract produced a pronounced and dose-dependent antinociceptive and anti-inflammatory effects in three above models. FJHQT extract after 28-day repeated administration did not caused any hematological, biochemical and histological change in rats. CONCLUSIONS: We suggest that FJHQT extract is a high safety index Chinese medicine for antinociceptive and anti-inflammatory application when Radix Stephania Tetrandra was correctly used in FJHQT. Its antinociceptive and anti-inflammatory mechanism might be related to peripheral nociceptive pathway such as prostaglandins.

Acute and subacute toxicity of the extract of Aristolochiae fructus and honey-fried Aristolochiae fructus in rodents.

Aristolochiae Fructus (AF) and honey-fried Aristolochiae Fructus (HAF) have been used in China for thousands of years as an anti-tussive and expectorant drug. Few clinical cases were reported associated with the toxicity of AF and HAF, although relatively high contents of aristolochic acids (AAs) were found in them. This work was designed to compare the acute and subacute toxicity of AF and HAF in order to provide references for safe clinical use and to evaluate the possibility of reducing toxicity of AF by honey-processing. The extracts of the herb were fed to mice or rats via gastric tube. Various toxic signs and symptoms, body weights, serum biochemical assay, organ weights and histopathology were used to evaluate the toxic effects. The median lethal dose (LD50) of AF and HAF are 34.1±7.2 g/kg/d and 62.6±8.0 g/kg/d with a 95% average trustable probability (p=0.95), respectively. The subacute results showed a dose-dependant relationship of the toxicity of AF and HAF. Even in the high dose groups, only moderate toxicity was observed. Honey-frying and decoction with water can decrease the contents of AAs, and attenuate the toxic effects of AF. But sufficient attention should be still paid to the safety of AF and HAF due to the existence of AAs.

[Advance in studies on toxicity of aristolochic acid and analysis on risk factors].

The renal toxicity and mutagenicity of aristolochic acid (AA) as well as its carcinogenicity on upper urinary tract transitional epithelial cells have been widely known. Since 2003, drug regulatory departments have successively cancelled the quality standards for AA-containing medicines such as Aristolochiae Radix, Aristolochiae Manshuriensis Caulis and Aristolchiae Fangchi Radix, and adopted measures for strengthening regulation and revising package insert or quality standards for other AA-containing medicines, including Aristolochia Cinnabarina Radix, Aristolochiae Fructus, Aristolochiae Mollissimae Herba, in order to control its safety risk. In recent years, domestic and foreign studies on AA have mainly involved action mechanism and clinical performance of AA toxicity, early-stage diagnosis and treatment method. In this paper, authors gave a brief summary and evaluation on risk factors for using AA-containing medicines, and offered measures and suggestions for preventing and controlling AA toxicity.

The safety of Homnawakod herbal formula containing Aristolochia tagala Cham. in Wistar rats.

BACKGROUND: A dried root of Aristolochia tagala Cham. (ATC) is often used in Thai traditional medicine as an antipyretic, anti-inflammatory agent, muscle relaxant, appetite-enhancing agent, and analeptic. Homnawakod, an important herbal recipe, originally contains ATC in its formula, however, some Aristolochia species have been reported to cause nephrotoxicity due to aristolochic acid (AA) and its derivatives, resulting in ATC removal from all formulae. Therefore, this study investigates the chemical profiles of ATC, the original (HNK+ATC) and the present Homnawakod Ayurved Siriraj Herbal Formulary™ (HNK), and investigates whether they could cause nephrotoxicity or aggravate LPS-induced organ injuries in vivo. METHODS: HPLC and LC/MS were used for chemical profile study. Male Wistar rats were randomly divided into groups in which the rats were intragastrically administered distilled water (2 groups), ATC (10 or 30 mg/kg), HNK+ATC (540 or 1,620 mg/kg), or HNK (1,590 mg/kg) for 21 days. A positive control group was administered with single dose 100 mg/kg standard AA-I intragastrically at day 1. Serum creatinine and urea were measured at baseline and at 7, 14 and 21 days of the treatment. On day 22, a model of lipopolysaccharide (LPS)-induced endotoxemia was used. One-way and two-way analyses of variance were performed and a P value of less than 0.05 was considered to be significant. RESULTS: The similarity of the HPLC chromatograms of HNK+ATC and HNK could suggest that the qualities of both formulae are nearly the same in terms of chemical profile. The amount of AA-I found in ATC is 0.24%w/w. All experimental groups exhibited similar levels of serum urea at baseline and 7 and 14 days of the treatment. At 21 days, rats received AA exhibited a significant increase in serum urea, whereas the others did not exhibit such toxicity. On day 22, there were no significant changes in LPS-induced renal and liver dysfunction, or LPS-induced mean arterial pressure (MAP) reduction upon administration of ATC, HNK+ATC, HNK or AA-I. CONCLUSIONS: These results suggest that ATC, HNK+ATC or HNK, at the animal dose equivalent to that used in human, do not cause the acute nephrotoxicity in rats and do not aggravate LPS-induced organ injuries even further.

Assessment of herbal medicinal products: challenges, and opportunities to increase the knowledge base for safety assessment.

Although herbal medicinal products (HMP) have been perceived by the public as relatively low risk, there has been more recognition of the potential risks associated with this type of product as the use of HMPs increases. Potential harm can occur via inherent toxicity of herbs, as well as from contamination, adulteration, plant misidentification, and interactions with other herbal products or pharmaceutical drugs. Regulatory safety assessment for HMPs relies on both the assessment of cases of adverse reactions and the review of published toxicity information. However, the conduct of such an integrated investigation has many challenges in terms of the quantity and quality of information. Adverse reactions are under-reported, product quality may be less than ideal, herbs have a complex composition and there is lack of information on the toxicity of medicinal herbs or their constituents. Nevertheless, opportunities exist to capitalise on newer information to increase the current body of scientific evidence. Novel sources of information are reviewed, such as the use of poison control data to augment adverse reaction information from national pharmacovigilance databases, and the use of more recent toxicological assessment techniques such as predictive toxicology and omics. The integration of all available information can reduce the uncertainty in decision making with respect to herbal medicinal products. The example of Aristolochia and aristolochic acids is used to highlight the challenges related to safety assessment, and the opportunities that exist to more accurately elucidate the toxicity of herbal medicines.

Toxicological risks of Chinese herbs.

As traditional Chinese medicine (TCM) has become more popular there have been increasing concerns about safety and potential toxicity of the Chinese materia medica (CMM) comprising plants, animal parts and minerals. The potential toxicity of many CMM is well recognised in TCM and to reduce risks use of some herbs is restricted whilst specific processing methods have been developed to modify the activities/toxicity of others. However adverse reactions have been reported, many of these are due misuse or abuse of Chinese medicine. The main problem remains products adulterated with pharmaceuticals for weight loss or erectile dysfunction. But some herbs have narrow therapeutic ranges (e.g., Aconitum species) so toxic effects are frequently reported. Toxic effects from chronic or cumulative dosing are difficult to detect in the traditional setting and recent reports have demonstrated the health problems from Aristolochia species. Despite safety concerns, Chinese medicine appears to be relatively safe with comparatively few reports of adverse reactions compared with overall drug reports. The wealth of information in the Chinese literature needs to be more widely available. As TCM is widely used by patients, improved pharmacovigilance and pharmacoepidemiology can contribute valuable safety information, relevant to clinical use.

Molecular markers in upper urothelial carcinoma associated to Balkan endemic nephropathy. Aristolochic acid as the major risk factor of the worldwide disease.

The role of aristolochic acid in the etiology of Balkan endemic nephropathy (BEN) and associated upper urothelial carcinoma (UUC) was recently confirmed. The aim of this study was to determine the marker(s) specific for BEN-associated UUC. A total of 82 patients with UUC (38 from the BEN region and 44 control tumors) were included in the study. The Ki-67 index in BEN tumors correlated with the grade and multifocality (p < 0.05), but in regression analysis, only the grade of BEN tumor. The p53 index was significantly higher in BEN than in control tumors (p < 0.05), as well as the alteration of p53 (p < 0.05). BEN low-stage tumors, tumors without limphovascular invasion (LVI), and tumors of the renal pelvis had a higher p53 index than the control tumors (p < 0.05, 0.01, 0.05, respectively). The Ki-67 index was higher in control tumors with high-stage and solid growth than in BEN UUC (p < 0.050, 0.005). The Ki-67 correlated with the grade, growth, stage, LVI, and multifocality of UUC on the best way, but not with the group. In regression analysis, only multifocality of UUC had predictive influence on Ki-67 activity (p < 0.001). P53 correlated with the grade, growth, and group (p < 0.05). This investigation identifies the p53 pathway as the specific cell cycle marker involved in BEN-associated UUC.

Aristolochic acid nephropathy: a worldwide problem.

Aristolochic acid nephropathy (AAN), a progressive renal interstitial fibrosis frequently associated with urothelial malignancies, was initially reported in a Belgian cohort of more than 100 patients after the intake of slimming pills containing a Chinese herb, Aristolochia fangchi. Although botanicals known or suspected to contain aristolochic acid (AA) were no longer permitted in many countries, several AAN cases were regularly observed all around the world. The incidence of AAN is probably much higher than initially thought, especially in Asia and the Balkans. In Asian countries, where traditional medicines are very popular, the complexity of the pharmacopoeia represents a high risk for AAN because of the frequent substitution of the botanical products by AA-containing herbs. In the Balkan regions, the exposure to AA found in flour obtained from wheat contaminated with seeds of Aristolochia clematitis could be responsible for the so-called Balkan-endemic nephropathy. Finally, despite the Food and Drug Administration's warnings concerning the safety of botanical remedies containing AA, these herbs are still sold via the Internet.

Role of environmental toxins in endemic (Balkan) nephropathy. October 2006, Zagreb, Croatia.

An international symposium, held in Zagreb, Croatia, in October 2006, brought together basic scientists and clinical investigators engaged in research on endemic (Balkan) nephropathy, a chronic renal tubulointerstitial disease of previously unknown cause that often is accompanied by upper urinary tract urothelial cancer. Although this disease is endemic in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia, a similar clinical entity occurs throughout Europe, Asia, and North America. Recent advances in the understanding of endemic nephropathy now favor the causative role of aristolochic acid over the ubiquitous mycotoxin known as ochratoxin A. Specifically, aristolactam-DNA adducts have been found in renal tissues and urothelial cancers of affected patients. A "signature" p53 mutation in the upper urothelial cancer associated with this disease provides evidence of long-term exposure to aristolochic acid. In addition, the renal pathophysiology and histopathology observed in endemic nephropathy most closely resemble the entity known as aristolochic acid nephropathy. Public health authorities in countries harboring this disease are encouraged to reduce the potential for dietary exposure to Aristolochia clematitis.

Omeprazole Alleviates Aristolochia manshuriensis Kom-Induced Acute Nephrotoxicity.

Aristolochia manshuriensis Kom (AMK) is a member of the Aristolochiaceae family and is a well-known cause of aristolochic acid (AA) nephropathy. In this study, we investigated the potential of omeprazole (OM) to alleviate AMK-induced nephrotoxicity. We found that OM reduced mouse mortality caused by AMK and attenuated AMK-induced acute nephrotoxicity in rats. OM enhanced hepatic Cyp 1a1/2 and renal Cyp 1a1 expression in rats, as well as CYP 1A1 expression in human renal tubular epithelial cells (HKCs). HKCs with ectopic CYP 1A1 expression were more tolerant to AA than the control cells. Therefore, OM may alleviate AMK-mediated acute nephrotoxicity through induction of CYP 1A1. We suggest that the coadministration of OM might be beneficial for reducing of AA-induced nephrotoxicity.

Substitution between Aristolochia and Bryonia genus in North-Eastern Morocco: toxicological implications.

ETHNOPHARMACOLOGICAL RELEVANCE: Although acknowledged as toxic herbs, Aristolochia species are still widely used worldwide. The aristolochic acids (AA) they contain can induce the so-called "aristolochic acid nephropathy", leading to renal fibrosis and upper urinary tract cancer. Traditional Moroccan medicine still often uses Aristolochia species under the vernacular name of Bereztem for the treatment of numerous ailments, notably cancer, diabetes or digestive tract disorders. As the botanical identity and renal toxicity of used species remain unexplored, the safety of patients may be threatened. MATERIAL AND METHODS: Ethnopharmacological data were collected from herbalists from the provinces of Oujda and Berkane, located in North-Eastern Morocco. Samples of Bereztem were collected at herbalist shops and checked for their content in AA using TLC and LC-MS methods. The toxicity of crude methanolic extracts of each herb was assessed on a HK-2 cell-based in vitro model by measurement of the cell survival to evaluate cytotoxicity and by assessment of renal-specific toxicity via (i) the evaluation of genes (E-cadherin and α-smooth muscle actin) expression by RT-qPCR; (ii) the quantities of ß-catenin and vimentin by immunofluorescence microscopy; (iii) the secretion of fibronectin; and (iv) the excretion of interleukin-6. RESULTS: The survey indicated that, among 42 herbalists visited, 33 were retailers of Bereztem, which was generally sold as a cancer treatment. Botanical investigations revealed that Aristolochia longa was frequently substituted by Bryonia dioica, which was associated with a higher cytotoxicity. Parameters specific to renal toxicity were also found to be enhanced, as compared to Aristolochia baetica and A. longa: down-regulation of ß-catenin and E-cadherin and up-regulation of vimentin and α-smooth muscle actin, and secretion of fibronectin and interleukin-6. CONCLUSION: In accordance with the Moroccan regulations, the use of so-called Aristolochia species should be discontinued. On one hand, the correctly identified aristolochia contain nephrotoxic aristolochic acids; on the other hand, aristolochia are massively substituted in North-Eastern Morocco and adulterated by a well-known toxic herb, B. dioica. Our data indicate that the bryony renal toxicity may be deleterious in shorter time periods than aristolochia. Reinforced on-site controls are needed to remind herbalists and harvesters that these herbs should be prohibited.

DNA adducts and p53 mutations in a patient with aristolochic acid-associated nephropathy.

BACKGROUND: Aristolochic acid-associated nephropathy (AAN) is a specific type of renal disease that predisposes patients to a high risk of urothelial carcinoma. The authors have analyzed DNA from a patient who had urothelial malignancy 6 years after presenting with AAN and later had a breast carcinoma that metastasized to the liver. METHODS AND RESULTS: DNA was isolated from the primary breast tumor, the liver tumor, and the original urothelial malignancy and assayed for aristolochic acid (AA)-DNA adducts and mutations in the p53 gene. The adduct detected was the adenosine adduct of aristolochic acid I 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI). In DNA from the breast and liver tumors the authors showed the same missense mutation in codon 245 (GGC-->GAC; Gly-->Asp) of exon 7 of p53. In contrast, DNA extracted from the urothelial tumor showed an AAG to TAG mutation in codon 139 (Lys-->Stop) of exon 5. CONCLUSION: A to T transversions, as observed here, are the typical mutations observed in the H-ras gene of tumors induced when rodents are treated with AA and correspond with DNA adduct formation at adenosine residues. These data indicate the probable molecular mechanism whereby AA causes urothelial malignancy.

Renal interstitial fibrosis and urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi).

A new renal disease called 'Chinese-herb nephropathy' (CHN) has been reported to occur in women who have ingested slimming pills containing powdered extracts of the Chinese herb Stephania tetrandra (ST). Moderate to end-stage renal disease developed, requiring renal replacement therapy by dialysis or transplantation. Phytochemical analyses of the pills revealed the presence of aristolochic acids (AA) instead of tetrandrine, suggesting the substitution of ST (Han fang ji) by Aristolochia fangchi containing nephrotoxic and carcinogenic AA. A typical histological feature of CHN is a progressive interstitial fibrosis leading to a severe atrophy of the proximal tubules, as documented by the urinary excretion rates of markers of tubular integrity (reduction of neutral endopeptidase enzymuria and high levels of microproteinurias). Removal of the native kidneys and ureters in end-stage CHN patients provided a high prevalence of urothelial carcinoma (46%). Tissue samples contained AA-related DNA adducts, which are not only specific markers of prior exposure to AA but are also directly involved in tumorigenesis. Exposure to Aristolochia species (spp.) is associated with the development of renal interstitial fibrosis (CHN) and urothelial cancer in humans. Health professionals should be aware that in traditional Chinese medicine, Aristolochia spp. are considered interchangeable with certain other herbal ingredients and are also sometimes mistaken for ST, Akebia, Asarum, Clematis spp. and Cocculus spp. in herbal remedies.

[Chinese herbs-induced renal failure with Fanconi syndrome: a report of 6 cases].

OBJECTIVE: To understand the clinical and pathological features of patients with Fanconi syndrome associated with renal function damage induced by Chinese herbs. METHODS: Six cases with herb-induced renal failure associated with Fanconi syndrome were clinicopathologically analyzed. RESULTS: All six patients had kidney insufficiency after ingestion of Chinese herbs. All of them took the herbs containing aristolochia manshuriensis definitely. Four of the six presented with rapidly progressive acute renal failure and one with acute renal failure. All of them had similar clinical features, such as polydipsia, polyuria, anemia, glycosuria, aminoaciduria, increased urine beta(2) microglobin (beta(2)m) excretion and proximal tubular acidosis. Renal biopsies performed in 3 cases showed extensive hypocellular interstitial fibrosis, tubular atrophy and loss of tubules. Glomeruli were apparently intact. CONCLUSIONS: Nephropathy caused by Chinese herbs may be associated with Fanconi syndrome and renal failure.

Fatal renal failure due to the Chinese herb "GuanMu Tong" (Aristolochia manshuriensis): autopsy findings and review of literature.

Herbal remedies have been used since ancient times and it is now known that they are not completely free of adverse effects. We present the case of a 41-year-old Chinese man, who died in renal failure because he consumed a herbal preparation called "Fen Qing Wu Lin Wan", having GuanMu Tong as main ingredient, for about 1 month. GuanMu Tong is derived from the plant Aristolochia manshuriensis which contains aristolochic acid. Aristolochic acid is being reported as the causative agent of what is now called aristolochic acid nephropathy (AAN) which includes Chinese herb nephropathy (CHN) and Balkan endemic nephropathy (BEN), all having renal impairment as hallmark for the disease. The gross autopsy showed multiple punctate hemorrhages over the limbs, pleural effusion, and edematous lungs with consolidation, mild myocardial hypertrophy and normal-looking kidneys. Microscopic renal tissue examination showed severe degeneration, necrosis and desquamation of renal tubular epithelial cells, presence of protein cast and a widened, edematous interstitium with interstitial fibrosis. We also provide the clinical presentation of the deceased as reported in the medical records and briefly review the literature pertinent to similar cases.