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.

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.

Ameliorative effect of cheqianzi decoction on hyperuricemia and kidney injury and underlying mechanism in rats.

Cheqianzi Decoction (CQD) is a Traditional Chinese Medicine (TCM) formula comprising four herbs and is recorded in the Ancient Materia Medica "Shengji Zonglu". Individually, these four herbs have been shown to reduce uric acid (UA) levels, to treat hyperuricemia (HUA), and alleviate kidney damage. However, the therapeutic efficacy of the CQD and related mechanism are not yet clear. In this study, high performance liquid chromatography (HPLC) analysis confirmed that the contents of the chemical components of the four herbal medicines were in accordance with the provisions of the Chinese Pharmacopoeia. A total of 99 potential targets were identified in the network pharmacology analysis of CQD, indicating its involvement in the regulation of inflammatory and apoptotic signaling pathways, and potential value for treating HUA and alleviating kidney injury. In vivo pharmacodynamic studies showed that compared with the Model group, significantly decreased levels of serum uric acid (SUA), serum creatinine (SCr), blood urea nitrogen (BUN) (all P < 0.05), and inflammatory factors (P < 0.01) were detected in the CQD group. Quantitative real-time PCR and Western blot analyses showed that compared with the Model group, adenosine triphosphate (ATP)-binding cassette efflux transporter G2 (ABCG2) expression in the CQD group was significantly upregulated (P < 0.01) at both the mRNA and protein levels, while mRNA expression of Caspase3 and NOD-like receptor family member 3 (NLRP3) (P < 0.05) and protein expression of NLRP3 (P < 0.01) were significantly downregulated. In conclusion, CQD promotes UA excretion by activating ABCG2, and induces inflammasome NLRP3-mediated reduction in inflammatory and apoptotic factors to achieve renal protection. Thus, our findings indicate the therapeutic potential of CQD in HUA with kidney injury.

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.

Differences in p38-STAT3-S100A11 signaling after the administration of aristolochic acid I and IVa may account for the disparity in their nephrotoxicity.

BACKGROUND: The safety of herbs containing aristolochic acids (AAs) has become a widespread concern. Previous reports indicate that AAs are highly nephrotoxic and carcinogenic, although there are more than 170 analogues of aristolochic acid. Not all AAs have the same degree of nephrotoxicity or carcinogenicity. Previous studies have found that aristolochic acid IVa (AA-IVa), the principal component of AAs within members of the Aristolochiaceae family, especially Asarum, a commonly used herb in China, has essentially no significant nephrotoxicity. However, several studies, including ours, have shown that aristolochic acid I (AA-I) is clearly nephrotoxic. PURPOSE: The focus of the study was to elucidate the molecular mechanism responsible for the difference in nephrotoxicity between the AA-I and AA-IVa. STUDY DESIGN/METHOD: Mice were administered with AA-I or AA-IVa for 22 weeks through the oral route, followed by a 50-week recovery time. The kidney tissues of mice were extracted at the end of 22 weeks. Pathological examination and proteomic detection (tandem mass tagging (TMT) and phosphorylated proteomics) were performed on the kidney tissue to investigate the key signaling pathways and targets of AAs-induced renal interstitial fibrosis (RIF). The key signaling pathways and targets were verified by Western blot (WB), siRNA transfection, and luciferase assays. RESULTS: AA-I caused severe nephrotoxicity, high mortality, and extensive RIF. However, the same AA-IVa dosage exhibited almost no nephrotoxicity and does not trigger RIF. The activation of the p38-STAT3-S100A11 signaling pathway and upregulated expression of α smooth muscle actin (α-SMA) and Bcl2-associated agonist of cell death (Bad) proteins could be the molecular mechanism underlying AA-I-induced nephrotoxicity. On the other hand, AA-IVa did not regulate the activation of the p38-STAT3-S100A11 signaling pathway and had relatively little effect on the expression of α-SMA and Bad. Consequently, the difference in the regulation of p38-STAT3-S100A11 pathway, α-SMA, and Bad proteins between AA-I and AA-IVa may be responsible for the divergence in their level of nephrotoxicity. CONCLUSION: This is the first study to reveal the molecular mechanism underlying the difference in nephrotoxicity between AA-I and AA-IVa. Whether STAT3 is activated or not may be the key factor leading to the difference in nephrotoxicity between AA-I and AA-IVa.

Studies on the metabolism and mechanism of acteoside in treating chronic glomerulonephritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Acteoside (ACT) is the main ingredient derived from the leaves of Rehmannia glutinosa (Dihuangye). Dihuangye has the function of clearing heat, replenishing qi and activating blood, nourishing yin and tonifying kidney in traditional Chinese medicine. Recent studies have demonstrated that Dihuangye can be used to treat nephritis and ACT is a promising antinephritic agent. AIM OF THE STUDY: To clarify the metabolites of ACT in biological samples and investigate the renoprotective effect and mechanism of ACT in rats with chronic glomerulonephritis (CGN). MATERIALS AND METHODS: In this study, the biotransformation of ACT in rat biological samples was clarified by quadrupole time-of-flight tandem mass spectrometry. The metabolites were validated by urine samples in nephropathy model rats. The effect of ACT and its metabolites was evaluated by glomerular podocyte injury due to high glucose. Based on an analysis of the ingredients in vivo, the potential therapeutic targets in the treatment of CGN were investigated by using network pharmacological analysis and molecular docking. Then, the renoprotective effect and mechanism of ACT were determined in rats in a passive Heymann nephritis (PHN) model. RESULTS: A total of 49 metabolites of ACT were detected and identified. Meanwhile, 21 metabolites were detected in nephropathy model rats. ACT was absorbed rapidly and transferred from the kidney, and the metabolites were eliminated via urine. The whole process lasted approximately 8 h. ACT had a significant protective effect on glomerular podocytes damaged by high glucose and 3,4-dihydroxyphenylacetic acid might be the main metabolite of ACT underlying its functions in vivo. The network pharmacology and molecular docking results showed 84 ACT-CGN targets, among which MAPK1, HRAS, AKT1, EGFR, and others were a highly correlated. In the PHN rat model, ACT significantly reduced the 24-h urine protein and serum creatinine concentrations, suppressed the leukocyte CD18 expression levels, decreased the serum tumor necrosis factor α (TNF-α) levels and tended to reduce serum interleukin 6 (IL-6) levels. ACT significantly reduced the platelet aggregation rate and inhibited the proliferative activity of splenic lymphocytes in response to the mitogen concanavalin A. Meanwhile, ACT inhibited transforming growth factor-ß and fibronectin expression in renal tissues and dose-dependently inhibited TNF-α and IL-6 production in RAW264.7 mouse macrophages at doses ranging from 1.8 to 1330 µg/mL. CONCLUSIONS: ACT had therapeutic effects on PHN rats, and its mechanism might be related to the inhibition of intercellular or intercellular-matrix adhesion, suppression of inflammatory response, regulation of immune function, improvement of tissue hemodynamics and hemorheology, and relief of fibrotic lesions.

[Risk assessment, safe medication and scientific supervision of traditional Chinese medicine containing aristolochic acids--toxicity is different among aristolochic acids, and detection and control of aristolochic acid â /â ¡ is critical].

The safety problem of traditional Chinese medicine containing aristolochic acid is of great concern in China and abraod, which poses a challenge in clinical application and supervision. There are many types of aristolochic acid analogues(AAAs) and 178 have been reported. According to the structure, they are classified into aristolochic acids(AAs) and aristololactams(ALs). The toxi-city is remarkably different among AAAs of different types. For example, AA-Ⅰ has strong nephrotoxicity and carcinogenicity, and the toxicity of AA-Ⅱ is lower than that of AA-Ⅰ. Besides, AA-Ⅳa and AA-Ⅰa are considered to have no obvious nephrotoxicity and carcinogenicity. The types and content of AAAs are significantly different among traditional Chinese medicines derived from different Aristolochiaceae species. For example, Asari Radix et Rhizoma and Aristolochiae Herba mainly consist of AAAs without obvious toxicity(such as AA-Ⅳa). The content of AAAs in compound preparations is related to the proportions of the medicinals and the processing method. The content of AA-Ⅰ in some compound preparations is very low or below the detection limit. Therefore, the author concludes that AAAs of different types have different toxicity, but not all AAAs has nephrotoxicity and carcinogenicity. Moreover, the toxicity of traditional Chinese medicines containing AAAs should not be generalized and AA-Ⅰ and AA-Ⅱ should be emphasized. In this paper, it is suggested that traditional Chinese medicine containing AAAs should be used rationally and research, analysis, and toxicological study of AAAs species and content should be strengthened. In addition, limit standards of AA-Ⅰ and AA-Ⅱ should be formulated and science-based supervision should be performed.

A review of the ethnopharmacology, phytochemistry, pharmacology and toxicology of Fructus Gardeniae (Zhi-zi).

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Gardeniae (FG) is the dried fruit of Gardenia jasminoides Ellis (GjE), which belongs to the family Rubiaceae. FG has a long history of use as a herb, and was originally recorded in Sheng Nong's herbal classic. FG has also been widely used as both medicine and food. AIM OF STUDY: This review aimed to provide a systematic and comprehensive analysis of the current research progress of FG in terms of ethnopharmacology, phytochemistry, pharmacology and toxicity, to provide new insights and extensive field of view for subsequent studies. METHODS: Scientific databases, including CNKI, VIP (Chinese literature), PubMed, Science Direct, Elsevier and Google Scholar (English literatures) were searched to gather data about FG and its main active ingredients such as geniposide and genipin (only regarding toxicity). RESULTS: Many chemical constituents have been identified from the fruit of GjE, including iridoids, terpenoids, flavonoids, organic acids, volatile oils and others. The constituents of different parts of FG and processed FG are different from those of whole FG. FG extract and its main active constituents have been reported to have pharmacological properties such as hepatoprotective, choleretic, anti-inflammatory, antioxidant, neuroprotective, anti-diabetic, anti-apoptotic and antitumor activities. However, an increasing number of studies have shown that FG induces multiple organ injury, especially causing hepatotoxicity and nephrotoxicity, which could increase the risk during clinical use. The available literature shows that geniposide, a major active component of FG and a critical marker for its quality, is associated with the pharmacology and toxicity of FG. CONCLUSION: Although a large number of studies examining FG have been published, issues remain. In the aspect of FG's pharmacology, the traditional efficacy and modern pharmacological effects of FG should be combined, which to broadens clinical application prospects. In addition, few studies have assessed the toxicity of FG. Toxicity assessment of FG should tackle various aspects, including compatibility, processing and the symptom-based prescription theory, in addition to over-dosage or long-term use, for a reasonable clinical use.

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.

αB-crystallin/HSPB2 is critical for hyperactive mTOR-induced cardiomyopathy.

Even though aberrant mechanistic target of rapamycin (mTOR) signaling is known to cause cardiomyopathy, its underlying mechanism remains poorly understood. Because augmentation of αB-crystallin and hspB2 was presented in the cortical tubers and lymphangioleiomyomatosis of tuberous sclerosis complex patients, we deciphered the role of αB-crystallin and its adjacent duplicate gene, hspB2, in hyperactive mTOR-induced cardiomyopathy. Cardiac Tsc1 deletion (T1-hKO) caused mouse mTOR activation and cardiomyopathy. Overexpression of αB-crystallin and hspB2 was presented in the hearts of these mice. Knockout of αB-crystallin/hspB2 reversed deficient Tsc1-mediated fetal gene expression, mTOR activation, mitochondrial damage, cardiomyocyte vacuolar degeneration, cardiomyocyte size, and fibrosis of T1-hKO mice. These cardiac-Tsc1; αB-crystallin; hspB2 triple knockout (tKO) mice had improved cardiac function, smaller heart weight to body weight ratio, and reduced lethality compared with T1-hKO mice. Even though activated mTOR suppressed autophagy in T1-hKO mice, ablation of αB-crystallin and hspB2 failed to restore autophagy in tKO mice. mTOR inhibitors suppressed αB-crystallin expression in T1-hKO mice and rat cardiomyocyte line H9C2. Starvation of H9C2 cells activated autophagy and suppressed αB-crystallin expression. Since inhibition of autophagy restored αB-crystallin expression in starved H9C2 cells, autophagy is a negative regulator of αB-crystallin expression. mTOR thus stimulates αB-crystallin expression through suppression of autophagy. In conclusion, αB-crystallin and hspB2 play a pivotal role in Tsc1 knockout-related cardiomyopathy and are therapeutic targets of hyperactive mTOR-associated cardiomyopathy.

Genome-wide transcriptional analysis of Aristolochia manshuriensis induced gastric carcinoma.

Context: Aristolochia manshuriensis Kom (Aristolochiaceae) (AMK) is known for toxicity and mutagenicity.Objective: The tumorigenic role of AMK has yet to be understood.Materials and methods: AMK extracts were extracted from root crude drug. SD (Sprague Dawley) rats underwent gavage with AMK (0.92 g/kg) every other day for 10 (AMK-10) or 20 (AMK-20) weeks. Stomach samples were gathered for histopathological evaluation, microarray and mRNA analysis.Results: The gastric weight to body weight ratio (GW/BW) is 1.7 in the AMK-10 cohort, and 1.8 in AMK-20 cohort compared to control (CTL) cohort. Liver function was damaged in AMK-10 and AMK-20 rats compared to CTL rats. There were no significant changes of CRE (creatinine) in AMK-10 and AMK-20 rats. Histopathological analysis revealed that rats developed dysplasia in the forestomach in AMK-10 rats, and became gastric carcinoma in AMK-20 rats. Genes including Mapk13, Nme1, Gsta4, Gstm1, Jun, Mgst2, Ggt6, Gpx2, Gpx8, Calml3, Rasgrp2, Cd44, Gsr, Dgkb, Rras, and Amt were found to be critical in AMK-10 and AMK-20 rats. Pik3cb, Plcb3, Tp53, Hras, Myc, Src, Akt1, Gnai3, and Fgfr3 worked in AMK-10 rats, and PDE2a and PDE3a played a pivotal role in AMK-20 rats.Discussion and conclusions: AMK induced benign or malignant gastric tumours depends on the period of AMK administration. Genes including Mapk13, Nme1, Gsta4, Gstm1, Jun, Mgst2, Ggt6, Gpx2, Gpx8, Calml3, Rasgrp2, Cd44, Gsr, Dgkb, Rras, Amt, Pik3cb, Plcb3, Tp53, Hras, Myc, Src, Akt1, Gnai3, Fgfr3, PDE2a, and PDE3a were found to be critical in aristolochic acid-induced gastric tumour process.

UAA and UAG may Encode Amino Acid in Cathepsin B Gene of Euplotes octocarinatus.

The ciliate Euplotes deviates from the universal genetic code by translating UGA as cysteine and using UAA and UAG as the termination codon. Here, we cloned and sequenced the Cathepsin B gene of Euplotes octocarinatus (Eo-CTSB) which containing several in-frame stop codons throughout the coding sequence. We provide evidences, based on 3'-RACE method and Western blot, that the Eo-CTSB gene is actively expressed. Comparison of the derived amino acid sequence with the homologs in other eukaryotes revealed that UAA and UAG may code for glutamine in Eo-CTSB. These findings imply an evolutionary complexity of stop codon reassignment in eukaryotes.

The Involvement of the RhoA/ROCK Signaling Pathway in Hypersensitivity Reactions Induced by Paclitaxel Injection.

A high incidence of hypersensitivity reactions (HSRs) largely limits the use of paclitaxel injection. Currently, these reactions are considered to be mediated by histamine release and complement activation. However, the evidence is insufficient and the molecular mechanism involved in paclitaxel injection-induced HSRs is still incompletely understood. In this study, a mice model mimicking vascular hyperpermeability was applied. The vascular leakage induced merely by excipients (polyoxyl 35 castor oil) was equivalent to the reactions evoked by paclitaxel injection under the same conditions. Treatment with paclitaxel injection could cause rapid histamine release. The vascular exudation was dramatically inhibited by pretreatment with a histamine antagonist. No significant change in paclitaxel injection-induced HSRs was observed in complement-deficient and complement-depleted mice. The RhoA/ROCK signaling pathway was activated by paclitaxel injection. Moreover, the ROCK inhibitor showed a protective effect on vascular leakage in the ears and on inflammation in the lungs. In conclusion, this study provided a suitable mice model for investigating the HSRs characterized by vascular hyperpermeability and confirmed the main sensitization of excipients in paclitaxel injection. Histamine release and RhoA/ROCK pathway activation, rather than complement activation, played an important role in paclitaxel injection-induced HSRs. Furthermore, the ROCK inhibitor may provide a potential preventive approach for paclitaxel injection side effects.

Systematic Overview of Aristolochic Acids: Nephrotoxicity, Carcinogenicity, and Underlying Mechanisms.

Aristolochic acids (AAs) are a group of toxins commonly present in the plants of genus Aristolochia and Asarum, which are spread all over the world. Since the 1990s, AA-induced nephropathy (AAN) and upper tract urothelial carcinoma (UTUC) have been reported in many countries. The underlying mechanisms of AAN and AA-induced UTUC have been extensively investigated. AA-derived DNA adducts are recognized as specific biomarkers of AA exposure, and a mutational signature predominantly characterized by A→T transversions has been detected in AA-induced UTUC tumor tissues. In addition, various enzymes and organic anion transporters are involved in AA-induced adverse reactions. The progressive lesions and mutational events initiated by AAs are irreversible, and no effective therapeutic regimen for AAN and AA-induced UTUC has been established until now. Because of several warnings on the toxic effects of AAs by the US Food and Drug Administration and the regulatory authorities of some other countries, the sale and use of AA-containing products have been banned or restricted in most countries. However, AA-related adverse events still occur, especially in the Asian and Balkan regions. Therefore, the use of AA-containing herbal remedies and the consumption of food contaminated by AAs still carry high risk. More strict precautions should be taken to protect the public from AA exposure.

Untargeted LC-MS-based metabonomics revealed that aristolochic acid I induces testicular toxicity by inhibiting amino acids metabolism, glucose metabolism, ß-oxidation of fatty acids and the TCA cycle in male mice.

As the main toxic component of aristolochic acid, aristolochic acid I (AAI) is primarily found in Aristolochiaceae plants such as Aristolochia, Aristolochia fangchi and Caulis aristolochiae manshuriensis. AAI has been proven to be carcinogenic, mutagenic and nephrotoxic. Although the role of AAI in testicular toxicity has been reported, its mechanism of action is unknown. Using metabonomics and molecular biology techniques, we tried to identify the differential endogenous metabolites of AAI that may affect the changes in testicular function in mice, map the network of metabolic pathways, and systematically reveal the molecular mechanism of AAI-induced testicular toxicity. We found that AAI inhibited amino acid metabolism in mouse testicular cells, impeded the uptake and oxidative decomposition of fatty acids, prevented normal glucose uptake by testicular cells, which inhibited glycolysis and gluconeogenesis, affected the mitochondrial tricarboxylic acid (TCA) cycle, which impaired the ATP energy supply, decreased the number of spermatogenic cells and sperm in the testes, induced changes in the mitochondrial state of spermatogonial cells, and ultimately led to physiological and pathological changes in the testes. AAI also regulated the testicular physiological activity by regulating the androgen receptor and hormone levels. This study used metabonomics and other methods to elucidate the mechanism of AAI-induced testicular toxicity from a new angle.

Research Advances and Prospects on Mechanism of Sinomenin on Histamine Release and the Binding to Histamine Receptors.

Sinomenine (SIN) is widely used in China to treat a variety of rheumatic diseases (RA), and has various pharmacological effects such as anti-inflammatory, analgesic, and anti-tumor effects. However, due to the histamine release characteristics of SIN, its adverse reactions such as allergic reactions, gastrointestinal reactions, and circulatory systemic reactions have been drawing increasing attention. We present here a systematic review of the chemical structure, pharmacological effects, clinical application, and adverse reactions of SIN, a detailed discussion on the relationship between histamine/histamine receptor and mechanism of action of SIN. In addition, we simulated the binding of SIN to four histamine receptors by using a virtual molecular docking method and found that the bonding intensity between SIN and receptors varied in the order shown as follows: H1R > H2R ~ H3R > H4R. The docking results suggested that SIN might exhibit dual regulatory effects in many processes such as cyclooxygenase-2 (COX-2) expression, NF-κB pathway activation, and degranulation of mast cells to release histamine, thereby exhibiting pro-inflammatory (adverse reactions)/anti-inflammatory effects. This study provides a theoretical basis for the clinical treatment of inflammations seen such as in RA using SIN, and also suggests that SIN has great potential in the field of cancer treatment and will have very important social and economic significance.

Binding of Organometallic Ruthenium Anticancer Complexes to DNA: Thermodynamic Base and Sequence Selectivity.

Organometallic ruthenium(II) complexes [(η6-arene)Ru(en)Cl][PF6] (arene = benzene (1), p-cymene (2), indane (3), and biphenyl (4); en = ethylenediamine) are promising anticancer drug candidates both in vitro and in vivo. In this paper, the interactions between ruthenium(II) complexes and 15-mer single- and double-stranded oligodeoxynucleotides (ODNs) were thermodynamically investigated using high performance liquid chromatography (HPLC) and electrospray ionization mass spectroscopy (ESI-MS). All of the complexes bind preferentially to G8 on the single strand 5'-CTCTCTT7G8T9CTTCTC-3' (I), with complex 4 containing the most hydrophobic ligand as the most reactive one. To the analogs of I (changing T7 and/or T9 to A and/or C), complex 4 shows a decreasing affinity to the G8 site in the following order: -AG8T- (K: 5.74 × 104 M-1) > -CG8C- > -TG8A- > -AG8A- > -AG8C- > -TG8T- (I) ≈ -CG8A- (K: 2.81 × 104 M-1). In the complementary strand of I, the G bases in the middle region are favored for ruthenation over guanine (G) bases in the end of oligodeoxynucleotides (ODNs). These results indicate that both the flanking bases (or base sequences) and the arene ligands play important roles in determining the binding preference, and the base- and sequence-selectivity, of ruthenium complex in binding to the ODNs.

Oral administration of Aristolochia manshuriensis Kom in rats induces tumors in multiple organs.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochia manshuriensis Kom (AMK), belonging to the Aristolochia family, is traditionally used in China to remove heart fire, promote dieresis, restore menstruation, and enhance milk secretion. The active constitutes of AMK are aristolochic acids (AAs, I and II) that are reported to cause serious side effects including nephrotoxicity and carcinogenicity. AIM OF THE STUDY: The tumorigenic role of AMK is far to be understood. We analyzed the toxicity reactions after long-term exposure of AMK in rats. MATERIALS AND METHODS: Sprague-Dawley rats underwent gavage with AMK doses of 51 mg/kg (AMK-1), 253 mg/kg (AMK-2), 508 mg/kg (AMK-3), 1029 mg/kg (AMK-4) or AAs of 15 mg/kg (AAs), and then sacrificed at the 6th, 10th, 14th, 18th, 22th, 26th and 30th weeks. Endpoint measurements included clinical observations, body weights, blood biochemistry, haematology and histomorphological observations. RESULTS: Body weight decreased after AMK or AAs treatment in rats. AMK destroyed renal function, and induced anemia in rats. AMK caused kidney, stomach, bladder and subcutaneous tumors in rats. In addition, primary hepatic carcinoma was not observed in rats. CONCLUSIONS: AMK had significant toxic effects in rats with regard to decreased body weight, diminished renal function, increased anemia and tumor incidence. Kidney, stomach, bladder and subcutaneous tissue are carcinogenic target organs of AMK or AAs, however liver is no- carcinogenic target organ of AMK or AAs in rats. AMK is carcinogenic in rats, and not be safe for humans.

miR-205 regulates the proliferation and invasion of ovarian cancer cells via suppressing PTEN/SMAD4 expression.

MicroRNAs (miRNAs/miRs) are non-coding RNAs that post-transcriptionally control target genes, and are involved in tumorigenesis, apoptosis, proliferation, invasion, metastasis and chemoresistance. However, data concerning miRNAs in ovarian cancer remain incomplete. The present study aimed to identify miRNAs that affected the malignant phenotype of ovarian cancer, and to analyze their potential mechanisms. The data demonstrated that miR-205 promoted cell proliferation and invasion of ovarian cancer cells via suppressing Phosphatase and tensin homolog (PTEN)/mothers against decapentaplegic homolog 4 (SMAD4) expression. Based on the Cancer Genome Atlas database analysis results, it was identified that miR-205 was significantly upregulated in ovarian cancer tissues and markedly correlated with poor prognosis in patients with ovarian cancer; its abnormal expression was also confirmed in tissues from patients with ovarian cancer by reverse transcription quantitative polymerase chain reaction. Additional Gene Ontology analysis revealed that the target genes of miR-205 were associated with cell proliferation and invasion. Consistent with the database analysis, miR-205 overexpression significantly promoted ovarian cancer cell proliferation and invasion in vitro. To additionally explore the mechanism by which miR-205 was associated with proliferation and invasion of ovarian cancer cells, a protein-protein interaction network was constructed based on miR-205 target genes associated with proliferation and invasion, and it was revealed that PTEN and SMAD4 were key target genes of miR-205. In ovarian cancer tissues, the expression levels of PTEN and SMAD4 were significantly downregulated, suggesting that miR-205 may suppress the expression of PTEN and SMAD4 in vivo. In vitro, miR-205 overexpression markedly suppressed the expression of SMAD4 and PTEN, additionally verifying that PTEN and SMAD4 were the target genes of miR-205 in ovarian cancer cells. These results elucidated the tumor-promoting role of miR-205 and established miR-205 as a potential treatment target for ovarian cancer.