Synergistic effects of Rapamycin and Fluorouracil to treat a gastric tumor in a PTEN conditional deletion mouse model.

The tumor suppressor gene phosphatase and tensin homolog (PTEN) in PI3K/Akt/mTOR pathway is essential in inhibiting tumor growth and metastasis. However, whether the mutation of PTEN gene could induce tumorigenesis and impact the treatment of gastric cancer is still unclear. The purpose of the study was to investigate the combined treatment of gastric tumorigenesis using Rapamycin and Fluorouracil (5-Fu) through interfering with the Akt/mTOR pathway in a mouse model with PTEN conditional deletion. Three groups of mice were exposed for 5 days to Rapamycin and 5-Fu separately and together. The gene expression of the Akt/mTOR pathway, the protein expression of caspase-3 and p-Akt, p-S6K and p-4EBP1, and the pathological changes in stomachs were analyzed. Our study demonstrates that the conditional PTEN deletion in the cells of glandular stomach induces hyperplastic gastric tumors in mice. The combined Rapamycin administration with 5-Fu resulted in better outcomes than their separate administration for the treatment of gastric cancer by inhibiting the mTOR signal pathway. Our study indicates that Rapamycin has a synergistic interaction with chemotherapeutic 5-Fu, and demonstrates a potential therapeutic combination treatment on glandular stomach tumor with PTEN functional absence or aberrantly activated Akt/mTOR pathway. It provides important insights into the inhibition of the Akt/mTOR pathway in gastric cancer clinical therapy.

Atorvastatin induces mitochondrial dysfunction and cell apoptosis in HepG2 cells via inhibition of the Nrf2 pathway.

Atorvastatin (ATO) is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor widely used to treat hypercholesterolemia. However, clinical application is limited by potential hepatotoxicity. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a master regulator of cellular antioxidants, and oxidative stress is implicated in statin-induced liver injury. This study investigated mechanisms of ATO-induced hepatotoxicity and potential mitigation by Nrf2 signaling. ATO reduced Nrf2 and antioxidant enzyme superoxide dismutase-2 (SOD2) expression in human hepatocarcinoma HepG2 cells. ATO also induced concentration-dependent HepG2 cell toxicity, reactive oxygen species (ROS) accumulation, and mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential (MMP) and cellular adenosine triphosphate (ATP). Further, ATO induced mitochondria-dependent apoptosis as indicated by increased Bax/Bcl-2 ratio, cleaved caspase-3, mitochondrial cytochrome c release and Annexin V-fluorescein isothiocyanate/propidium iodide staining. Tert-butylhydroquinone enhanced Nrf2 and SOD2 expression, and partially reversed ATO-induced cytotoxicity, ROS accumulation, MMP reduction, ATP depletion and mitochondria-dependent apoptosis. In conclusion, the present study demonstrates that ATO induces mitochondrial dysfunction and cell apoptosis in HepG2 cells, at least in part, via inhibition of the Nrf2 pathway. Nrf2 pathway activation is a potential prevention for ATO-induced liver injury.

Subchronic Oral Toxicity Evaluation of Lanthanum: A 90-day, Repeated Dose Study in Rats.

OBJECTIVE: The present study was undertaken to evaluate the subchronic toxicity of lanthanum and to determine the no observed adverse effect level (NOAEL), which is a critical factor in the establishment of an acceptable dietary intake (ADI). METHODS: In accordance with the Organization for Economic Co-operation and Development (OECD) testing guidelines, lanthanum nitrate was administered once daily by gavage to Sprague-Dawley (SD) rats at dose levels of 0, 1.5, 6.0, 24.0, and 144.0 mg/kg body weight (BW) per day for 90 days, followed by a recovery period of 4 weeks in the 144.0 mg/kg BW per day and normal control groups. Outcome parameters were mortality, clinical symptoms, body and organ weights, serum chemistry, and food consumption, as well as ophthalmic, urinary, hematologic, and histopathologic indicators. The benchmark dose (BMD) approach was applied to estimate a point of departure for the hazard risk assessment of lanthanum. RESULTS: Significant decreases were found in the 144.0 mg/kg BW group in the growth index, including body weight, organ weights, and food consumption. This study suggests that the NOAEL of lanthanum nitrate is 24.0 mg/kg BW per day. Importantly, the 95% lower confidence value of the benchmark dose (BMDL) was estimated as 9.4 mg/kg BW per day in females and 19.3 mg/kg BW per day in males. CONCLUSION: The present subchronic oral exposure toxicity study may provide scientific data for the risk assessment of lanthanum and other rare earth elements (REEs).

Subchronic toxicity study of yttrium nitrate by 90-day repeated oral exposure in rats.

Concerns regarding the adverse effects of long-term exposure to low levels of rare earth elements (REEs) from foods on human health have arisen in recent years. Nevertheless, no official acceptable daily intake (ADI) has yet been proposed for either total REEs or individual REE. In accordance with the Organization for Economic Co-operation and Development (OECD) testing guideline, the present study was undertaken to evaluate the subchronic toxicity of yttrium, a representative heavy REE with higher contaminated level in foods in China, to achieve a no observed adverse effect level (NOAEL) which is a critical basis for the establishment of an ADI. Yttrium nitrate was orally administered to rats at doses of 0, 10, 30 and 90 mg/kg/day for 90 days followed by a recovery period of 4 weeks. The following toxicity indices were measured: mortality, clinical signs, daily food consumption and weekly body weight; urinalysis, hematology, blood coagulation, clinical biochemistry and histopathology at the end of administration and recovery periods. No toxicologically significant changes were found in any yttrium-treated group as compared to the concurrent control group. Under the present experimental condition, the NOAEL in rats was thus set at 90 mg/kg for yttrium nitrate, i.e. 29.1 mg/kg for yttrium.

AMPK activator acadesine fails to alleviate isoniazid-caused mitochondrial instability in HepG2 cells.

Isoniazid (INH) is a first-line antituberculosis drug that is adversely associated with hepatotoxicity. Recently, impairment of mitochondrial homeostasis involved in this side effect has been noticed. Mitochondrial homeostasis is achieved by the balance between the generation of functional mitochondria by biogenesis and elimination of dysfunctional mitochondria by autophagy. AMP-activated protein kinase (AMPK) can maintain mitochondrial stability through positive control of these two processes. In this study, we showed that AMPK activator acadesine (AICAR) alleviated INH-caused impairment of mitochondrial biogenesis by activation of silent information regulator two ortholog 1 (SIRT1)-peroxisome proliferator-activated receptor γ coactivator 1α (PGC1 α) pathway in HepG2 cells. However, mitochondrial instability and apoptosis were caused by AICAR along with an unexpected decrease in INH-induced cytoprotective autophagy. Therefore, AICAR failed to alleviate INH-caused mitochondrial instability in HepG2 cells due to its inhibitory effect on autophagy induced by INH. Copyright © 2017 John Wiley & Sons, Ltd.

[Development of human embryonic stem cell platforms for human health-safety evaluation].

The human embryonic stem cells (hESCs) serve as a self-renewable, genetically-healthy, pluripotent and single source of all body cells, tissues and organs. Therefore, it is considered as the good standard for all human stem cells by US, Europe and international authorities. In this study, the standard and healthy human mesenchymal progenitors, ligament tissues, cardiomyocytes, keratinocytes, primary neurons, fibroblasts, and salivary serous cells were differentiated from hESCs. The human cellular health-safety of NaF, retinoic acid, 5-fluorouracil, dexamethasone, penicillin G, adriamycin, lead acetate PbAc, bisphenol A-biglycidyl methacrylate (Bis-GMA) were evaluated selectively on the standardized platforms of hESCs, hESCs-derived cardiomyocytes, keratinocytes, primary neurons, and fibroblasts. The evaluations were compared with those on the currently most adopted cellular platforms. Particularly, the sensitivity difference of PM2.5 toxicity on standardized and healthy hESCs derived fibroblasts, currently adopted immortalized human bronchial epithelial cells Beas-2B and human umbilical vein endothelial cells (HUVECs) were evaluated. The RESULTS showed that the standardized hESCs cellular platforms provided more sensitivity and accuracy for human cellular health-safety evaluation.

Combined exposure to nano-silica and lead induced potentiation of oxidative stress and DNA damage in human lung epithelial cells.

Growing evidence has confirmed that exposure to ambient particulate matters (PM) is associated with increased morbidity and mortality of cardiovascular and pulmonary diseases. Ambient PM is a complex mixture of particles and air pollutants. Harmful effects of PM are specifically associated with ultrafine particles (UFPs) that can adsorb high concentrations of toxic air pollutants and are easily inhaled into the lungs. However, combined effects of UFPs and air pollutants on human health remain unclear. In the present study, we elucidated the combined toxicity of silica nanoparticles (nano-SiO2), a typical UFP, and lead acetate (Pb), a typical air pollutant. Lung adenocarcinoma A549 cells were exposed to nano-SiO2 and Pb alone or their combination, and their combined toxicity was investigated by focusing on cellular oxidative stress and DNA damage. Factorial analyses were performed to determine the potential interactions between nano-SiO2 and Pb. Our results showed that exposure of A549 cells to a modest cytotoxic concentration of Pb alone induced oxidative stress, as evidenced by elevated reactive oxygen species generation and lipid peroxidation, and reduced glutathione content and superoxide dismutase and glutathione peroxidase activities. In addition, exposure of A549 cells to Pb alone induced DNA damage, as evaluated by alkaline comet assay. Exposure of A549 cells to non-cytotoxic concentration of nano-SiO2 did not induce cellular oxidative stress and DNA damage. However, exposure to the combination of nano-SiO2 and Pb potentiated oxidative stress and DNA damage in A549 cells. Factorial analyses indicated that the potentiation of combined toxicity of nano-SiO2 and Pb was induced by additive or synergistic interactions.

[Development of human embryonic stem cell model for toxicity evaluation].

The current international standard for toxicity screening of biomedical devices and materials recommend the use of immortalized cell lines because of their homogeneous morphologies and infinite proliferation which provide good reproducibility for in vitro cytotoxicity screening. However, most of the widely used immortalized cell lines are derived from animals and may not be representative of normal human cell behavior in vivo, in particular in terms of the cytotoxic and genotoxic response. Therefore, It is vital to develop a model for toxicity evaluation. In our studies, two Chinese human embryonic stem cell (hESC) lines as toxicity model were established. hESC derived tissue/organ cell model for tissue/organ specific toxicity evaluation were developed. The efficiency and accuracy of using hESC model for cytoxicity, embryotoxicity and genotoxicity evaluation were confirmed. The results indicated that hESCs might be good tools for toxicity testing and biosafety evaluation in vitro.

[Effects of metallothionein on rifampicin (RFP)-induced hepatotoxicity in mice].

OBJECTIVE: To investigate the effect of metallothionein (MT) on rifampicin (RFP)-induced hepatotoxicity and the possible mechanisms. METHODS: Male MT- I / II null (MT-/-) and wild type (MT+/+) mice were randomly divided into 4 groups, respectively, and were orally administered RFP (50, 100 or 200 mg/kg) or equal volumes of solvent daily for 15 consecutive days. Levels of plasma alanine aminotransferase (ALT), alkaline phosphatase (ALP) and direct bilirubin (DB) were determined. Liver indexes were calculated and liver histopathologic changes were examined by hematoxylin and eosin (HE) staining. The content of glutathione (GSH) as well as the activities of glutathione peroxydase (GPx) and glutathione reductases (GR) were measured in the liver homogenates. RESULTS: RFP treatment induced significant increases in plasma ALT, AST and DB, as well as liver index. Significant histopathologic changes which were charactered as fatty degeneration in liver were noteced. Moreover, augmentations of GSH content and GR activity and attenuation of GPx activity were observed. More severe hepatic injuries in MT-/- mice were observed as compared to MT+/+ mice, which were evidenced by higher liver/body weight ratio and GR activity, lower GSH content and GPx activity, and more serious fatty degeneration. CONCLUSION: RFP-induced hepatotoxicity was associated with cholestasis and oxidative damage. MT -/- mice were more susceptible than MT +/+ mice to RFP-induced hepatotoxicity and the enhanced hepatotoxicity involves increased oxidative stress.

Basal expression of metallothionein suppresses butenolide-induced oxidative stress in liver homogenates in vitro.

Butenolide (4-acetamido-4-hydroxy-2-butenoic acid gamma-lactone) is a Fusarium mycotoxin which is frequently detected in foodstuffs and feedstuffs for human and animal consumption. It can evoke a broad spectrum of toxicities, thus posing a potential health risk to both humans and animals. Previous study showed that this mycotoxin produced a significant oxidative stress, and several antioxidants abated this effect. Metallothionein (MT) has been proposed as a potent antioxidant, therefore, this study attempts to determine whether endogenous expression of MT protects against butenolide-induced hepatic oxidative stress by using an in vitro incubation system of liver homogenates prepared from MT-I/II null (MT-/-) mice, and the corresponding wild type (MT+/+) mice. The results showed that butenolide elicited significant oxidative stress in both MT-/- mice and MT+/+ mice; however, MT-/- mice were more sensitive than MT+/+ mice to butenolide-induced hepatic oxidative stress, as evidenced by more production of thiobarbituric acid reactive substances and nitric oxide, and by more severe reductions of glutathione, superoxide dismutase and glutathione peroxidase in the liver homogenates of MT-/- mice than those of MT+/+ mice. These findings implicated the antioxidant potency of basal expression of MT in suppression of the oxidative stress of butenolide.

Decreased aortic contractile reaction to 5-hydroxytryptamine in rats with long-term hypertension induced by lead (Pb(2+)) exposure.

Male Wistar rats were exposed to 100ppm Pb(2+) in drinking water for 10 months. Tail blood pressure, serum 5-hydroxytryptamine (5-HT), the expression of 5-HT(2B) receptor in the aorta, the aortic response to 5-HT, and the pathologic changes of aorta were examined. The systolic blood pressure of Pb(2+) exposed group was significantly increased after 2 months of Pb(2+) exposure. After 10 months of Pb(2+) exposure, aortic contractile response to 5-HT was significantly decreased. There was no significant difference in the levels of serum 5-HT and the expression of 5-HT(2B) receptor between these two groups. The aortic media and the media-lumen ratio of Pb(2+) exposed group were significantly increased. These data suggest that long-term Pb(2+) exposure can increase blood pressure, and can alter the function and structure of aortic of rats. The decreased aortic response to 5-HT has little relation to the expression of 5-HT(2B) receptor and the serum level of 5-HT, maybe is a result of the aortic structural alteration.

Chlorpyrifos induces delayed cytotoxicity after withdrawal in primary hippocampal neurons through extracellular signal-regulated kinase inhibition.

In this study, the delayed effect and related mechanism after chlorpyrifos (CPF) withdrawal was studied in primary rat hippocampal neurons. The results showed that 10 muM CPF induced no detectable cytotoxicity during 96 h continuous exposure while its withdrawal after 48 h exposure induced evident cytotoxicity, as indexed by decreased methyl thiazolyl tetrazolium (MTT) metabolism, increased loss of neurons immunostained by neuron-specific enolase (NSE) antibody, and the increased terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) positive cell rate in the following 24 h and 48 h incubation in the absence of CPF. Extracellular signal-related kinase (ERK)1/2 activation by phosphorylation was observed and persisted during CPF exposure. However, CPF withdrawal after 48 h exposure led to inhibition of ERK1/2 phosphorylation. Carbacol and nerve growth factor (NGF), which are ERK1/2 activators, protected the neurons after CPF withdrawal, while atropine and PD98059, which are ERK1/2 inhibitors, exacerbated the cytotoxicity, indicating the involvement of inhibition of ERK1/2 phosphorylation in CPF-induced delayed cytotoxicity. In conclusion, CPF withdrawal after exposure induced delayed cytotoxicity in cultured neurons. Inhibition of ERK1/2 phosphorylation was found to be related to the delayed cytotoxicity. This finding may provide a new insight into the toxicological mechanism of organophosphorus pesticides, especially chronic organophosphate-induced neuropsychiatric disorder characterized by delayed occurrence.

Chronic organophosphate (OP)-induced neuropsychiatric disorder is a withdrawal syndrome.

Chronic organophosphate-induced neuropsychiatric disorder is a less well-characterized syndrome, which is usually delay-occurred, persists long and is similar to the symptom of cholinergic deficit, its mechanism is unclear. The characteristics of chronic organophosphate-induced neuropsychiatric disorder are somewhat opposite to the direct action of OP pesticide, since withdrawal effect is usually opposite to the original effect of a drug, hypothesis that chronic organophosphate-induced neuropsychiatric disorder is a kind of withdrawal syndrome is suggested.

Combined effects of repeated administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin and polychlorinated biphenyls on kidneys of male rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs) are persistent environmental contaminants that exist as complex mixtures in the environment, but the possible interactions of TCDD and PCBs have not been systematically investigated. The main objective of this study was to investigate the combined nephrotoxic effects of TCDD and PCBs on rats and to reveal the potential interactions between TCDD and PCBs. Male Sprague-Dawley rats were intragastrically administered TCDD (10 microg/kg), PCBs (Aroclor 1254, 10 mg/kg), or the combination (10 microg/kg TCDD + 10 mg/kg Aroclor 1254). After 12 consecutive days of exposure, all treatments induced nephrotoxicity, as evidenced by significant increases in the levels of serum creatinine and blood urea nitrogen, changes of kidney histopathology, and significant renal oxidative stress. Most of these effects were more remarkable in the combined-exposure group. Furthermore, all treatments induced renal cytochrome P450 1A1 (CYP1A1) protein expression, and the induction was more conspicuous in the combined-exposure group. These findings suggested that the nephrotoxicity induced by TCDD and PCBs in the present study might be attributable to the high expression of CYP1A1. In addition, the result of the two-way analysis of variance revealed that the combined effects of TCDD and PCBs were complicated, being additive, synergistic, or antagonistic depending on the selection of toxicity end points under the present experimental condition. This study demonstrates that combined exposure to TCDD and PCBs induced significant nephrotoxicity in rats, and there were complicated interactions between the two pollutants on the nephrotoxicity.

Effects of Fusarium mycotoxin butenolide on myocardial mitochondria in vitro.

Fusarium mycotoxin toxicosis has been implicated in the etiology of Keshan disease, an endemic mitochondrial cardiomyopathy prevailing in certain areas of China. Butenolide (4-acetamido-4-hydroxy-2-butenoic acid gamma-lactone) is one of the Fusarium mycotoxins which are frequently detected from cereal grains in endemic areas. A recent study indicates that this mycotoxin induces rat cardiotoxicity, but its effect on the myocardial mitochondria remains unclear. The present study is therefore undertaken to explore the toxic effect potential of butenolide on the myocardial mitochondria. Exposure of cultured cardiac myocytes to 50 microg/ml of butenolide provoked dissipation of mitochondrial membrane potential. Incubation of isolated rat myocardial mitochondria with butenolide of 100 microg/ml for 60 min resulted in mitochondrial swelling, indicating the occurrence of mitochondrial permeability transition. Furthermore, marked oxidative damage in myocardial mitochondria was observed after incubation of isolated myocardial mitochondria with butenolide ranging from 0 to 50 microg/ml for 60 min, as manifested by concentration-dependent increases in the production of thiobarbituric acid reactive substances, the indicator of lipid peroxidation. Contrarily, a representative antioxidant glutathione significantly alleviated this oxidative mitochondrial damage induced by butenolide. In conclusion, these observations clearly show that butenolide can induce dysfunction of myocardial mitochondria, and oxidative damage appears to play a crucial role in these deleterious effects. The present study supports the hypothesis that mycotoxin toxicosis is a probable etiological factor of Keshan disease, the mitochondrial cardiomyopathy.

[Gene expression profile of isoniazid liver-injured rat using cDNA microarray].

OBJECTIVE: To investigate the changes of gene expression profile of rat liver tissue by cDNA microarrays. METHODS: Twenty Wistar rats in control group (n = 10) and isoniazid (INH) group (n = 10) were orally administrated with normal saline and 400 mg/kg INH for 14 days, respectively. The differentially expressed genes significantly correlated with liver injury were screened and analyzed. The mechanisms of liver injury caused by INH were specifically analyzed at level of gene expression based on the biological functions of those differentially expressed genes. RESULTS: Thirty-seven differentially expressed genes were found in the rats administrated with INH. Among them, 25 genes were up-regulated, while the other 12 genes were down-regulated. These differentially expressed genes were functionally related to the changes of CYP450-related genes, fatty acid metabolism, and protein metabolism. CONCLUSION: INH can cause the remarkable changes of the gene expression profiles in rat liver cells, which is important for further elucidating the mechanisms of liver injury caused by INH.

[Nephrotoxicity study of Aristolochia fangchi in rats by metabonomics].

OBJECTIVE: To study the changes of metabolites in rat urine after treatment of Aristolochia fangchi decoction by metabonomic method. METHODS: Sixty-four male SD rats were divided into Aristolochia fangchi group and normal control group. Rats in the Aristolochia fangchi group were orally administered with 8.1 g/(kg.d) of Aristolochia fangchi and the normal control group was administered with equal volume of distilled water for 4 weeks. Twenty-four hour urine was collected at different time points (before, after 2- and 4-week administration and 2 weeks after administration) and their H nuclear magnetic resonance (NMR) spectra were acquired and subjected to data process, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) or orthogonal signal correction (OSC). The contents of blood urea nitrogen (BUN) and serum creatinine (SCr) and histopathological changes of the renal tissues were also detected. RESULTS: The content of BUN of the Aristolochia fangchi group was markedly higher than that of the normal control group after 2-week administration (P<0.05), and cellular edema in tubular endothelial cells, structure damage of glomeruli and inflammatory cell infiltration were found in the kidney. Along with the lasting of administration to 4-week, the renal injury in the Aristolochia fangchi group became more serious, and the contents of BUN and SCr were all significantly higher as compared with the normal control group (P<0.05). Two weeks after administration, the content of BUN in the Aristolochia fangchi group was still higher than that in the normal control group (P<0.05), and the pathological changes in renal tissues were not different from those on the 4th week. The urine of the Aristolochia fangchi group could be readily discriminated from the normal control group at every time point based on PCA. During the whole administration period, compared with the normal control group, the concentration of urinary taurine was increased time-dependently, while the citrate was decreased in the Aristolochia fangchi group. The concentration of hippurate was increased at the 2nd week and the 6th week (2 weeks after drug withdrawal) but decreased at the 4th week; the concentration of creatinine/creatine was increased at the 4th week but decreased at the 6th week; the concentration of 2-oxo-glutarate was decreased and the concentration of trimethylamine oxide was increased at the 4th and 6th week. CONCLUSION: High-dose Aristolochia fangchi can induce renal lesion and its seriousness is correspondent to the lasting of administration. Aristolochia fangchi may also have toxicity on liver.

Lipid peroxidation and antioxidant defense impairment in the hearts of chick embryos induced by in ovo exposure to Fusarium mycotoxin butenolide.

Fusarium mycotoxin toxicosis has been implicated as an etiological factor for Keshan disease, an endemic cardiomyopathy prevailing in specific regions of China. Butenolide (4-acetamido-4-hydroxy-2-butenoic acid gamma-lactone) is one of the Fusarium mycotoxins frequently detected from the foodstuffs of endemic areas and has been shown to possess the potential to induce cardiotoxicity, implying this mycotoxin might play a role in the occurrence of Keshan disease. The present study was undertaken to further investigate the myocardial toxicity of butenolide from a viewpoint of oxidative damage. A single in ovo injection of butenolide to chick embryos, an excellent in vitro toxicology model resulted in significant oxidative injuries to the myocardium, manifested by a dose-dependent depletion of sulfhydryl groups, reduction of glutathione peroxidase (GPx) activity, and increase of thiobarbituric acid reactive substances production, a hallmark of lipid peroxidation. In contrast, co-injections of butenolide to chick embryos with sodium selenite or vitamin C, two potent antioxidants, markedly abated these oxidative injuries. In conclusion, the present study clearly indicated that butenolide could induce significant myocardial oxidative injuries. The current findings reinforce the hypothesis that toxicosis by Fusarium mycotoxins may be one of the etiological factors for Keshan disease, and oxidative damage is involved in the pathogenesis of myocardial toxicity.

[Metabonomic profile of urine from rats administrated with different treatment period of rifampin].

OBJECTIVE: To study the effect of rifampin (RFP) on the metabonomic profile of rat urine and its relationship with traditional toxicity evaluation of blood biochemical indicators and histopathology. METHODS: Thirty-six male Wistar rats were randomly divided into control group, 50 mg/kg RFP group, and 100 mg/kg RFP group, with 12 rats in each group. Rats in each group were given intragastric infusion with a daily dose of 0, 50 mg/kg RFP, and 100 mg/kg RFP for 3, 7, and 14 days, respectively. Then 4 rats in each group were killed on the next day of administration to collect blood samples and liver sample for the determination of blood biochemical indicators and for the pathological analysis of the liver. The urine specimens over 24 hours of each rat were collected before and after each treatment until the rat was killed. 1H nuclear magnetic resonance (1H NMR) spectra of these urine specimens were acquired and subjected to data preprocess and principal component analyses (PCA). RESULTS: The level of serum total bilirubin of the rat administrated with 100 mg/(kg x d) RFP for 7 days was significantly higher than that of control group (P < 0.05). Mild hepatotoxicity to the rat, treated with RFP of higher dosage (100 mg/kg) and longer duration (14 days), was revealed by the traditional histopathological method. The metabonomic spectra of rat urine in different groups differed from each other; a trajectory bias in determination of rat urine by 1H NMR occurred depending on the administration duration. As demonstrated by 1H NMR spectra of urine in rats treated with RFP, the concentration of urinary citrate and 2-oxoglutarate decreased, along with the remarkable increase of the concentrations of urinary taurine and glucose (compared with those of the control group). CONCLUSIONS: Being consistent with the results of traditional toxicity evaluation measurements, metabonomic method is more sensitive. The 1H-NMR metabonomic profile of the rat urine is closely related with the duration of RFP. The hepatic toxicity induced by RFP is related to the reduction of energy metabolism in tricarboxylic acid cycle and the perturbation of glucose and lipid metabolism.