Study on the mechanism of cantharidin-induced hepatotoxicity in rat using serum and liver metabolomics combined with conventional pathology methods

Cantharidin (CTD), a compound secreted from Mylabris species, exhibits strong antitumor properties; however, hepatotoxicity restricts its clinical application. The mechanism by which CTD induces toxicity remains unclear. In the present study, the hepatotoxicity of CTD in the rat was investigated using a metabolomic approach combined with conventional pathology methods. A total of 30 rats were intragastrically treated with two doses of CTD (0.75 and 1.5 mg/kg) for 15 days to evaluate hepatotoxicity. Serum and liver samples were collected for biochemical dynamics analyses, histopathological examination and metabolomic analysis. It was found that liver index and serum biochemical indices were significantly increased. Furthermore, the pathology results showed that hepatocytes and subcellular organelles were damaged. Metabolomics analysis found 4 biomarkers in serum and 15 in the liver that were associated with CTD-induced hepatotoxicity. In addition, these were responsible for CTD hepatotoxicity by glycerophospholipid metabolism, sphingolipid metabolism, and steroid hormone biosynthesis. In conclusion, conventional pathology and metabolomics for exploring hepatotoxicity can provide useful information about the safety and potential risks of CTD.     

Cantharidin-induced LO2 cell autophagy and apoptosis via endoplasmic reticulum stress pathway in vitro

Cantharidin (CTD), an important active compound derived from the traditional Chinese medicine Mylabris (also called Banmao), has been used in the treatment of diseases such as tumors and dermatosis. However, Mylabris has been shown to induce hepatotoxicity in clinical practice and animal experiments, limiting its use. Further, a detailed mechanism underlying CTD-induced hepatotoxicity has not been determined. In the present study, we aimed to explore the effect of endoplasmic reticulum stress (ERS), autophagy, and apoptosis on CTD-induced hepatotoxicity. We found that CTD could inhibit the proliferation of LO2 cells; increase alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and malondialdehyde levels; and reduce glutathione peroxidase and superoxide dismutase activities. Western blotting showed that low concentrations of CTD induced the expressions of ERS-related proteins [GRP78, ATF4, PERK, p-PERK, XBP1–1 s, and CHOP], but high concentrations of CTD inhibited their expressions. Furthermore, high concentrations of CTD activated autophagy (LC3, Beclin-1, Atg3, Atg4A, Atg4B, and Atg7), induced the expressions of apoptotic proteins (Bax/Bcl-2 and caspase-3), and increased LO2 toxicity. Taken together, these results indicated that CTD can induce LO2 cytotoxicity by inhibiting ERS and inducing autophagy and apoptosis, which provides a scientific basis for CTD-induced hepatotoxicity.     

Discovery of common urinary biomarkers for hepatotoxicity induced by carbon tetrachloride, acetaminophen and methotrexate by mass spectrometry-based metabolomics

Liver toxicity represents an important healthcare issue because it causes significant morbidity and mortality and can be difficult to predict before symptoms appear owing to drug therapy or exposure to toxicants. Using metabolomic techniques, we discovered common biomarkers for the prediction of hepatotoxicity in rat urine using mass spectrometry. For this purpose, liver toxicity was induced by 5 days of oral administration of carbon tetrachloride (1 ml kg^?1 per day), acetaminophen (1000 mg kg^?1 per day) and methotrexate (50 mg kg^?1 per day). Serum levels of alkaline phosphatase aspartate aminotransferase, alanine aminotransferase and histopathology in liver tissue were then checked to demonstrate liver toxicity. Global metabolic profiling with UPLC‐TOF‐MS (ultraperformance liquid chromatography–mass spectrometry), multivariate analysis (partial least square‐discriminant analysis, hierarchical analysis) and database searching were performed to discover common biomarkers for liver toxicity induced by these three compounds. Urinary concentrations of the newly discovered biomarkers were then quantified to confirm them as biomarkers of hepatotoxicity with targeted metabolic profiling using GC (gas chromatography)–MS and CE (capillary electrophoresis)–MS. In the results, steroids, amino acids and bile acids were metabolically changed between the control and drug‐treated groups in global metabolic profiling; 11β‐hydroxyandrosterone, epiandrosterone, estrone, 11‐dehydrocorticosterone, glycine, alanine, valine, leucine, dl ‐ornithine, 3‐methylhistidine, cholic acid and lithocholic acid were selected as liver toxicity biomarkers after performing targeted metabolic profiling. In conclusion, we discovered metabolite biomarkers belonging to three different metabolic pathways to check for liver toxicity with mass spectrometry from a metabolomics study that could be used to evaluate hepatotoxicity induced by drugs or other toxic compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous characterization of bile acids and their sulfate metabolites in mouse liver, plasma, bile, and urine using LC-MS/MS

Sulfation is a major metabolic pathway involved in the elimination and detoxification of bile acids (BAs). Several lines of evidence are available to support the role of sulfation as a defensive mechanism to attenuate the toxicity of accumulated BAs during hepatobiliary diseases. Individual BAs and their sulfate metabolites vary markedly in their physiological roles as well as their toxicities. Therefore, analytical techniques are required for the quantification of individual BAs and BA-sulfates in biological fluids and tissues. Here we report a simple, sensitive, and validated LC-MS/MS method for the simultaneous quantification of major BAs and BA-sulfates in mouse liver, plasma, bile, and urine. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for liver and plasma) was used to extract BAs and BA-sulfates. Base-line separation of all analytes (unsulfated- and sulfated BAs) was achieved in 25min with a limit of quantification of 1ng/ml. This LC-MS/MS method was applied to simultaneously quantify BAs and BA-sulfates in both male and female mouse tissues and fluids. Less than 3% of total BAs are present in the sulfate form in the mouse liver, plasma, and bile, which provides strong evidence that sulfation is a minor metabolic pathway of BA elimination and detoxification in mice. Furthermore, we report that the marked female-predominant expression of Sult2a1 is not reflected into a female-predominant pattern of BA-sulfation.     

Species differences in bile acids II. Bile acid metabolism

One of the mechanisms of drug‐induced liver injury (DILI) involves alterations in bile acid (BA) homeostasis and elimination, which encompass several metabolic pathways including hydroxylation, amidation, sulfation, glucuronidation and glutathione conjugation. Species differences in BA metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably the DILI during the early stages of drug discovery and development. We developed an in vitro cofactor‐fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs (cholic acid, chenodeoxycholic acid, lithocholic acid and ursodeoxycholic acid) between humans and several animal species. High‐ and low‐resolution liquid chromatography–tandem mass spectrometry and nuclear magnetic resonance imaging were used for the qualitative and quantitative analysis of BAs and their metabolites. Major species differences were found in the metabolism of BAs. Sulfation into 3‐O‐sulfates was a major pathway in human and chimpanzee (4.8%–52%) and it was a minor pathway in all other species (0.02%–14%). Amidation was primarily with glycine (62%–95%) in minipig and rabbit and it was primarily with taurine (43%–81%) in human, chimpanzee, dog, hamster, rat and mice. Hydroxylation was highest (13%–80%) in rat and mice followed by hamster, while it was lowest (1.6%–22%) in human, chimpanzee and minipig. C6‐β hydroxylation was predominant (65%–95%) in rat and mice, while it was at C6‐α position in minipig (36%–97%). Glucuronidation was highest in dog (10%–56%), while it was a minor pathway in all other species (<12%). The relative contribution of the various pathways involved in BA metabolism in vitro were in agreement with the observed plasma and urinary BA profiles in vivo and were able to predict and quantify the species differences in BA metabolism. In general, overall, BA metabolism in chimpanzee is most similar to human, while BA metabolism in rats and mice is most dissimilar from human.     

Acetaminophen-induced hepatotoxicity in rats is correlated with the accumulation of bile acids: an underlying mechanism

In the present study, we aimed to investigate the underlying mechanism of acetaminophen (APAP)-induced hepatotoxicityby measuring the expression levels of liver transporters and concentrations of bile acids (BAs) in rat plasma and liver. SD rats (42)were randomly assigned into six groups, including 6-h control group, APAP 6-h group, 12-h control group, APAP 12-h group, 24-h control group and APAP 24-h group. The estimation study of BAs in plasma and liver was performed on LC-MS/MS.The levels of bile salt export pump (Bsep), multidrug resistant protein 2 (Mrp2), multidrug resistant protein 4 (Mrp4), Na⁺/taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 2 (Oatp2) in the liver were analyzed by Western blotting analysis. Compared with the corresponding control groups, no difference was found in the BA levels and the expressions of BA transporters in the plasma and liver after 6 h of APAP administration. While BA levels were significantly decreased in the plasma and increased in the liver after 12 h of APAP administration (P<0.05); and the expressions of Bsep and Mrp2 were significantly reduced (P<0.05). After 24 h of APAP administration, BA levels were both greatly increased in the plasma and liver (P<0.05); and the expressions of Mrp4 and Oatp2 were significantly decreased (P<0.05). In response to over-dose APAP, Bsep, Mrp2, Mrp4 and Oatp2 levels were reduced at different time points, causing the accumulation of BAs, and such accumulation may ultimately lead to the severe liver injury, which could be an underlying mechanism of the APAP-induced hepatotoxicity.     

Increased bile acids in enterohepatic circulation by short-term calorie restriction in male mice

Previous studies showed glucose and insulin signaling can regulate bile acid (BA) metabolism during fasting or feeding. However, limited knowledge is available on the effect of calorie restriction (CR), a well-known anti-aging intervention, on BA homeostasis. To address this, the present study utilized a “dose–response” model of CR, where male C57BL/6 mice were fed 0, 15, 30, or 40% CR diets for one month, followed by BA profiling in various compartments of the enterohepatic circulation by UPLC-MS/MS technique. This study showed that 40% CR increased the BA pool size (162%) as well as total BAs in serum, gallbladder, and small intestinal contents. In addition, CR “dose-dependently” increased the concentrations of tauro-cholic acid (TCA) and many secondary BAs (produced by intestinal bacteria) in serum, such as tauro-deoxycholic acid (TDCA), DCA, lithocholic acid, ω-muricholic acid (ωMCA), and hyodeoxycholic acid. Notably, 40% CR increased TDCA by over 1000% (serum, liver, and gallbladder). Interestingly, 40% CR increased the proportion of 12α-hydroxylated BAs (CA and DCA), which correlated with improved glucose tolerance and lipid parameters. The CR-induced increase in BAs correlated with increased expression of BA-synthetic (Cyp7a1) and conjugating enzymes (BAL), and the ileal BA-binding protein (Ibabp). These results suggest that CR increases BAs in male mice possibly through orchestrated increases in BA synthesis and conjugation in liver as well as intracellular transport in ileum.     

Toxicity and intracellular accumulation of bile acids in sandwich-cultured rat hepatocytes: Role of glycine conjugates

Excessive intrahepatic accumulation of bile acids (BAs) is a key mechanism underlying cholestasis. The aim of this study was to quantitatively explore the relationship between cytotoxicity of BAs and their intracellular accumulation in sandwich-cultured rat hepatocytes (SCRH). Following exposure of SCRH (on day-1 after seeding) to various BAs for 24 h, glycine-conjugated BAs were most potent in exerting toxicity. Moreover, unconjugated BAs showed significantly higher toxicity in day-1 compared to day-3 SCRH. When day-1/-3 SCRH were exposed (0.5–4 h) to 5–100 μM (C)DCA, intracellular levels of unconjugated (C)DCA were similar, while intracellular levels of glycine conjugates were up to 4-fold lower in day-3 compared to day-1 SCRH. Sinusoidal efflux was by far the predominant efflux pathway of conjugated BAs both in day-1 and day-3 SCRH, while canalicular BA efflux showed substantial interbatch variability. After 4 h exposure to (C)DCA, intracellular glycine conjugate levels were at least 10-fold higher than taurine conjugate levels. Taken together, reduced BA conjugate formation in day-3 SCRH results in lower intracellular glycine conjugate concentrations, explaining decreased toxicity of (C)DCA in day-3 versus day-1 SCRH. Our data provide for the first time a direct link between BA toxicity and glycine conjugate exposure in SCRH.     

vMAlteration of bile acid metabolism in pseudo germ-free rats

To characterize the impact of gut microbiota on host bile acid metabolism, we investigated the metabolic profiles of oxysterols and bile acids (BAs) in a conventional rat model (SD) (n=5) and its pseudo germ-free (GF) equivalent (n=5). GF rats were developed by the oral administration of bacitracin, neomycin and streptomycin (200 mg/kg, each) twice a day for 6 days. Urinary levels of oxysterols and bile acid metabolites were quantified using gas chromatography-mass spectrometry (GC-MS). The activity levels of enzymes involved in the bile acid metabolic pathway were determined through urinary concentration ratio between product to precursor. Cholic acid (CA) and ??-/??-muricholic acid (??-/??-MCA) were significantly elevated at pseudo germ-free condition. An increase of hydroxylase (cholesterol 7??-hydroxylase, oxysterol 7??-hydroxylase and cytochrome P450 scc) and a significant decrease of 7??-dehydroxylase were observed. The urinary concentration ratio of primary bile acids, a marker for hepatotoxicity, increased in pseudo germfree conditions. Therefore, it was found that gut microbiota could play a significant role in the bile acids homeostasis and metabolism.     

内质网应激、自噬与凋亡在斑蝥素致大鼠肝毒性中的作用

目的 探讨斑蝥素(cantharidin，CTD)致大鼠药物性肝损伤(drug-induced liver injury，DILI)的毒理学机制。方法 采用不同剂量CTD(0.061 4，0.092 1，0.184 1 mg·kg⁻¹)连续灌胃SD大鼠 28 d，检测肝脏指数和血清肝功能指标，HE 染色评估肝脏病理变化。进一步采用免疫印迹法检测内质网应激(endoplasmic reticulum stress，ERS)、自噬和细胞凋亡通路蛋白。结果 CTD 干预后肝脏指数显著升高，生化指标ALT、AST、LDH、ALP和T-Bil显著升高， 且呈剂量依赖性，肝脏组织出现结构破坏和中央静脉扩张等病理变化；GRP78、CHOP、ATF4、Beclin-1、LC3、Caspase-3、Caspase-8和Bax/Bcl-2的蛋白表达水平显著升高。分子对接结果显示，GRP78、ATF4和Beclin-1与CTD对接结果良好。结论 CTD可激活大鼠ERS，进一步激活自噬，诱导下游凋亡，研究结果可为CTD诱导的DILI提供新的科学依据。     

Diet supplementation with cholic acid promotes intestinal epithelial proliferation in rats exposed to γ-radiation

Consumption of a high-fat diet increases some secondary bile acids (BAs) such as deoxycholic acid (DCA) in feces. DCA is derived from cholic acid (CA), a primary BA. We evaluated intestinal epithelial proliferation and BA metabolism in response to oral administration of cholic acid (CA) in rats to determine the influence of a CA diet on the responses of gut epithelia to γ-rays. WKAH/HkmSlc rats were divided into two dietary groups: control diet or CA-supplemented (2 g/kg diet) diet. Some of the rats from each group were irradiated with γ-rays, and epithelial cell proliferation in the colon was analyzed histochemically. Unirradiated CA-fed rats had high levels of DCA and CA in the sera, as well as the presence of taurocholic acid in their feces. Significant increases were observed in both epithelial proliferation and the number of epithelial cells in the colon of the CA-fed rats, and this effect was observed at 8 weeks after γ-ray exposure. Furthermore, extracts from both cecal contents and sera of the unirradiated CA-fed rats promoted proliferation of IEC-6 cells. These results indicate that BAs in enterohepatic circulation promote proliferation and survival of the intestinal epithelium after receiving DNA damage.     

Quantitative relationship between bile acid structure and biliary lipid secretion in rats

A series of unconjugated and taurine conjugated bile acids (BAs) differing in water solubility (SWo), critical micellar concentration (CMC), and hydrophilicity (K') were infused iv to rats at a tracer dose and a dose of 6 mumol/min/kg over a 1-h period. Bile was collected for 3 h to evaluate the role of BA structure on cholesterol, phospholipids secretions, and bile flow. The BAs studied differ in the number (2-3), position (-3, -6, -7, -12), and orientation of the hydroxyls (alpha/beta); the side chain structure was modified by shortening (C-23, nor-BA) and by lengthening (C-25, homo-BA), while maintaining the same structure of nuclear hydroxyls (3 alpha 7 beta). At a "tracer" dose, all C-24 natural BAs are efficiently recovered in bile when administered in both unconjugated and taurine conjugated forms. At a "high dose", all taurine conjugated BAs are efficiently recovered in bile (80-100%). However, a variable recovery was observed among unconjugated BAs: trihydroxy BAs are efficiently recovered (85-100%), while dihydroxy BAs are only partially recovered (25-40%). The side chain-modified BAs [i.e., C-23 nor and C-25 homo analogs of ursodeoxycholic acid (UDCA)] are partially recovered at a tracer dose (20-30%), but less at a high dose (10-20%) when administered in the unconjugated form. In contrast, the corresponding taurine conjugates are more efficiently recovered in bile (60-80%). Conjugation with taurine increases total recovery of unconjugated BAs in bile by not more than 30-40%. Highly hydrophilic and water-soluble BAs, such as ursocholic acid (SWo = 1.67 mM) and cholic acid (SWo = 0.27 mM), can also be secreted as unconjugates, and this accounts for their complete recovery. The conjugation step is rate limiting for poorly soluble BAs such as ursodeoxycholic acid (SWo = 0.009 mM) when administered at a high dose, and critical for nor and homo analogs which are poorly soluble and whose side chain modification partially suppresses their conjugation with taurine or glycine and thereby induces alternative pathways such as glucuronidation or sulfation. The induced bile flow is directly related to the hydrophilicity of the natural C-24 bile acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Species differences in bile acids I. Plasma and urine bile acid composition

Maintenance of bile acid (BA) homeostasis is essential to achieve their physiologic functions and avoid their toxic effects. The marked differences in BA composition between preclinical safety models and humans may play a major role in the poor prediction of drug‐induced liver injury using preclinical models. We compared the composition of plasma and urinary BAs and their metabolites between humans and several animal species. Total BA pools and their composition varied widely among different species. Highest sulfation of BAs was observed in human and chimpanzee. Glycine amidation was predominant in human, minipig, hamster and rabbit, while taurine amidation was predominant in mice, rat and dogs. BA profiles consisted primarily of tri‐OH BAs in hamster, rat, dog and mice, di‐OH BAs in human, rabbit and minipig, and mono‐OH BA in chimpanzee. BA profiles comprised primarily hydrophilic and less toxic BAs in mice, rat, pig and hamster, while it primarily comprised hydrophobic and more toxic BAs in human, rabbit and chimpanzee. Therefore, the hydrophobicity index was lowest in minipig and mice, while it was highest in rabbit, monkey and human. Glucuronidation and glutathione conjugation were low in all species across all BAs. Total concentration of BAs in urine was up to 10× higher and more hydrophilic than plasma in most species. This was due to the presence of more tri‐OH, amidated, sulfated and primary BAs, in urine compared to plasma. In general, BA profiles of chimpanzee and monkeys were most similar to human, while minipig, rat and mice were most dissimilar to human.     

In vitro bile acid-dependent hepatocyte toxicity assay system using human induced pluripotent stem cell-derived hepatocytes: Current status and disadvantages to overcome

Cholestatic drug-induced liver injury (DILI) is a type of hepatotoxicity. Its underlying mechanisms are dysfunction of bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4), which play major roles in bile acid (BA) excretion into the bile canaliculi and blood, resulting in accumulation of BAs in hepatocytes. The sandwich-cultured hepatocyte (SCH) model can simultaneously analyze hepatic uptake and biliary excretion. Therefore, we investigated whether sandwich-cultured human induced pluripotent stem cell (iPS cell)-derived hepatocytes (SCHiHs) are suitable for evaluating cholestatic DILI. Fluorescent N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2′-aminoethanesulfonate (tauro-nor-THCA-24-DBD, a BSEP substrate) was accumulated in bile canaliculi, which supports the presence of a functional bile canaliculi lumen. MRP2 was highly expressed in the Western blot analysis, whereas the mRNA expression of BSEP was hardly detectable. MRP3/4 mRNA levels were maintained. Of the 22 compounds known to cause DILI with BAs, 7 showed significant cytotoxicity. Most high-risk drugs were detected using the developed SCHiH system. However, a shortcoming was the considerably low expression level of BSEP, which prevented the detection of some relevant drugs whose risks should be detected in primary human hepatocytes.     

Cadmium-induced apoptosis of hepatocytes is not associated with death receptor-related caspase-dependent pathways in the rat

Cadmium (Cd) is a heavy metal of considerable environmental and occupational concern. The liver is the major target organ of Cd toxicity that follows from repeated exposure to Cd. The aim of this study was to investigate the mechanism of cell death of Cd-induced hepatotoxicity in a rat model. Eighteen adult male Sprague–Dawley (SD) rats were injected daily with a dose of Cd acetate (30 μM/kg body weight, subcutaneously). After 1, 2 and 7 days rats were euthanized and blood and liver tissues were sampled for analysis. Biochemical analyses of the level of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were undertaken. Histopathological and Western blot analyses for liver cellular damage and cell death were also performed. The results for the Cd-treated group of animals were compared to those from 12 control rats. The serum AST/ALT levels increased significantly 24 h after CD exposure. From the Western blot analyses, activation of Bid, independent of caspase-8 was seen and Bax induced the release of cytochrome c into the cytosol from mitochondria in a dose-dependent manner. The level of Bcl-2 was decreased. Eventually, caspase-9 and caspase-3 were activated, and poly(ADP-ribose) polymerase (PARP) was cleaved in a dose-dependent manner. A histopathological analysis and DNA fragmentation test showed apoptotic cell death of the hepatocytes increased over time. These results suggest that Cd-induced liver cell apoptosis in the rat, over a period of 7 days, may not be related to the death-receptor pathway. Moreover, apoptosis is dose-dependent and associated with the decrement of Bcl-2.     

Hepatocyte-based in vitro model for assessment of drug-induced cholestasis

Early detection of drug-induced cholestasis remains a challenge during drug development. We have developed and validated a biorelevant sandwich-cultured hepatocytes- (SCH) based model that can identify compounds causing cholestasis by altering bile acid disposition. Human and rat SCH were exposed (24–48 h) to known cholestatic and/or hepatotoxic compounds, in the presence or in the absence of a concentrated mixture of bile acids (BAs). Urea assay was used to assess (compromised) hepatocyte functionality at the end of the incubations. The cholestatic potential of the compounds was expressed by calculating a drug-induced cholestasis index (DICI), reflecting the relative residual urea formation by hepatocytes co-incubated with BAs and test compound as compared to hepatocytes treated with test compound alone. Compounds with clinical reports of cholestasis, including cyclosporin A, troglitazone, chlorpromazine, bosentan, ticlopidine, ritonavir, and midecamycin showed enhanced toxicity in the presence of BAs (DICI ≤ 0.8) for at least one of the tested concentrations. In contrast, the in vitro toxicity of compounds causing hepatotoxicity by other mechanisms (including diclofenac, valproic acid, amiodarone and acetaminophen), remained unchanged in the presence of BAs. A safety margin (SM) for drug-induced cholestasis was calculated as the ratio of lowest in vitro concentration for which was DICI ≤ 0.8, to the reported mean peak therapeutic plasma concentration. SM values obtained in human SCH correlated well with reported % incidence of clinical drug-induced cholestasis, while no correlation was observed in rat SCH. This in vitro model enables early identification of drug candidates causing cholestasis by disturbed BA handling.     

Anlotinib improves bile duct ligature-induced liver fibrosis in rats via antiangiogenesis regulated by VEGFR2/mTOR pathway

Cholestasis is a main clinical feature of biliary atresia (BA), which leads to liver fibrosis (LF). The focus of BA treatment is preventing and slowing the progress of LF. This study reports the improvement effect of anlotinib on common bile duct ligature (BDL)-induced LF in young rats. The BDL young rats were treated with anlotinib and the serum levels of aspartate aminotransferase, alanine aminotransferase, albumin, and total bilirubin were determined. Histological staining was performed and pathological changes in liver tissue were observed. The expression levels of α-SMA, collagen I, CD31, TGF-β1, phospho-VEGFR2, phospho-4E/BP1, and phospho-S6K1 were determined. The results showed that anlotinib significantly improved the liver function and histopathological injury of BDL rats, inhibited the deposition of collagen and hepatocyte apoptosis, and downregulated the protein expression of α-SMA and collagen I. Furthermore, anlotinib treatment significantly inhibited microvascular formation in the liver and downregulated the expression level of phospho-VEGFR2, thereby suggesting that the antifibrosis effect of anlotinib may be achieved by antiangiogenesis. In addition, anlotinib downregulated the expression of phospho-S6K1 and upregulated the expression of phospho-4E/BP1, two downstream proteins of the mammalian target of rapamycin (mTOR) pathway. MHY1485, an agonist of mTOR, significantly reversed the inhibitory effect of anlotinib on angiogenesis and LF but did not influence the effect of anlotinib on the downregulation of phospho-VEGFR2 expression. Together, the above-mentioned results suggest that the effect of anlotinib on BDL-induced LF involves at least antiangiogenesis regulated by the VEGFR2/mTOR signaling pathway.     

Gas chromatography–mass spectrometry‐based profiling of serum fatty acids in acetaminophen‐induced liver injured rats

In this study, we have developed and validated a simple, accurate and sensitive gas chromatography–mass spectrometry (GC‐MS) method for simultaneous quantification of 18 fatty acids in rat serum, including both non‐esterified (NEFA) and esterified (EFA) fatty acids, and subsequent analysis of fatty acid metabolic profiles. This novel method was used to evaluate the serum levels of fatty acids from vehicle‐ and acetaminophen (APAP)‐treated rats. Serum levels of 7 NEFAs and 14 EFAs were significantly higher in APAP‐treated rats 24 h after APAP administration at 1500 mg kg^–1 when compared with vehicle‐treated controls. Control and APAP‐treated rats could be differentiated based on their metabolic profiles using two different chemometric analysis methods: principle component analysis (PCA) and partial least squares‐discriminant analysis (PLS‐DA). More importantly, we identified the following NEFAs as potential biomarkers of APAP‐induced liver injury: oleic acid (C18:1n9), linoleic acid (C18:2n6), docosahexaenoic acid (C22:6n3) and arachidonic acid (C20:4n6). The serum concentrations of C18:1n9, C18:2n6 and C22:6n3 were all positively correlated (r > 0.8; Pearson's correlation analysis) with the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). These results suggest that a novel targeted metabolomics method based on the metabolic profiling of fatty acids analyzed by GC‐MS provides exact serum concentrations of fatty acids as well as a prospective methodology to evaluate chemically induced hepatotoxicity. Copyright © 2012 John Wiley & Sons, Ltd.     

GC-TOF/MS-based metabolomic profiling of estrogen deficiency-induced obesity in ovariectomized rats

Aim:

To explore the alteration of endogenous metabolites and identify potential biomarkers using metabolomic profiling with gas chromatography coupled a time-of-flight mass analyzer (GC/TOF-MS) in a rat model of estrogen-deficiency-induced obesity.

Methods:

Twelve female Sprague-Dawley rats six month of age were either sham-operated or ovariectomized (OVX). Rat blood was collected, and serum was analyzed for biomarkers using standard colorimetric methods with commercial assay kits and a metabolomic approach with GC/TOF-MS. The data were analyzed using multivariate statistical techniques.

Results:

A high body weight and body mass index inversely correlated with serum estradiol (E2) in the OVX rats compared to the sham rats. Estrogen deficiency also significantly increased serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol. Utilizing GC/TOF-MS-based metabolomic analysis and the partial least-squares discriminant analysis, the OVX samples were discriminated from the shams. Elevated levels of cholesterol, glycerol, glucose, arachidonic acid, glutamic acid, glycine, and cystine and reduced alanine levels were observed. Serum glucose metabolism, energy metabolism, lipid metabolism, and amino acid metabolism were involved in estrogen-deficiency-induced obesity in OVX rats.

Conclusion:

The series of potential biomarkers identified in the present study provided fingerprints of rat metabolomic changes during obesity and an overview of multiple metabolic pathways during the progression of obesity involving glucose metabolism, lipid metabolism, and amino acid metabolism.     

Identification of quinone imine containing glutathione conjugates of diclofenac in rat bile

High-resolution accurate MS with an LTQ-Orbitrap was used to identify quinone imine metabolites derived from the 5-hydroxy (5-OH) and 4 prime-hydroxy (4'-OH) glutathione conjugates of diclofenac in rat bile. The initial quinone imine metabolites formed by oxidation of diclofenac have been postulated to be reactive intermediates potentially involved in diclofenac-mediated hepatotoxicity; while these metabolites could be formed using in vitro systems, they have never been detected in vivo. This report describes the identification of secondary quinone imine metabolites derived from 5-OH and 4'-OH diclofenac glutathione conjugates in rat bile. To verify the proposed structures, the diclofenac quinone imine GSH conjugate standards were prepared synthetically and enzymatically. The novel metabolite peaks displayed the identical retention times, accurate mass MS/MS spectra, and the fragmentation patterns as the corresponding authentic standards. The formation of these secondary quinone metabolites occurs only under conditions where bile salt homeostasis was experimentally altered. Standard practice in biliary excretion experiments using bile duct-cannulated rats includes infusion of taurocholic acid and/or other bile acids to replace those lost due to continuous collection of bile; for this experiment, the rats received no replacement bile acid infusion. High-resolution accurate mass spectrometry data and comparison with chemically and enzymatically prepared quinone imines of diclofenac glutathione conjugates support the identification of these metabolites. A mechanism for the formation of these reactive quinone imine containing glutathione conjugates of diclofenac is proposed.