Liver Tissue Extracts Research Articles

Poly chlorinated dibenzo- p-dioxins and dibenzofurans in different tissues of the cormorants ( Phalacrocorax carbo) from Dongting Lake, China

Tissue distribution provides important information regarding the pharmacokinetic behavior of pollutants and is invaluable when analyzing the risk posed to avian species by the exposure to such kind of pollutants. In this study, concentrations of polychlorinated dibenzo- p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were determined in muscle, liver, spleen, kidney, stomach, gall bladder, skin, heart, pancreas, intestine and lung tissue extracts of cormorants collected from Dongting Lake, China. Tissue distribution results showed preferential accumulation of PCDD/Fs in both liver and skin. The total concentration of PCDD/Fs ranged from 421 to 5696 pg/g lipid weight. Octachlorinated dibenzo- p-dioxin (OCDD) was the predominant congener in all tissues and contributed between 31% and 82% to all 17 PCDD/Fs in different tissues. The liver/muscle ratios progressively increased with the degree of chlorination of PCDDs, except for OCDD. The relative toxic potential of PCDDs and PCDFs in tissues were calculated using the World Health Organization (WHO) Toxic Equivalency Factors (TEFs) for birds. The concentrations of WHO-toxic equivalent in different tissues ranged between 14.8 and 2021 pg/g lipid weight. These results indicated PCDD/Fs may have been bio-accumulated in cormorant via food-web. Furthermore, when compared with studies reported in the literatures, the PCDD/Fs levels in the cormorant collected from Dongting Lake were still relatively high.

Metabolomics characterization of energy metabolism reveals glycogen accumulation in gut-microbiota-lacking mice

Microbiota in the gut are considered an important environmental factor associated with host metabolism and physiology. Although gut microbiota are known to contribute to hepatic lipogenesis and fat storage, little is known about how the condition influences the deposition of glycogen in the liver. To better understand and characterize the host energy metabolism in guts lacking microbiota, we compared the liver metabolome of specific pathogen-free and germ-free mice by gas chromatography–mass spectrometry combined with partial least-squares discriminant analysis. We identified 30 of 52 highly reproducible peaks in chromatograms of liver tissue extracts from the two groups of mice. The two groups showed significant differences in metabolic profile. Changes in liver metabolism involved metabolites such as amino acids, fatty acids, organic acids and carbohydrates. The metabolic profile of germ-free mice suggests that they synthesize glycogen and accumulate it in the liver through gluconeogenesis and glycogenesis. Our findings shed light on a new perspective of the role of gut microbiota in energy metabolism and will be useful to help study probiotics, obesity and metabolic diseases.

NMR‐based metabonomic approach on the toxicological effects of a Cimicifuga triterpenoid

Cimicifugae Rhizoma, a well-known botanical dietary supplement, has been the subject of intense interest due to its potential application for alleviating menopausal symptom. Although there are clinic data that the Cimicifuga extract should have hepatotoxicity, no evidence on the main chemical components has been reported. Cimicidol-3-O-β -d-xyloside (CX) is one of the main triterpenoids of the rhizome. This work studies the toxicological effects of CX after oral administration (50 mg kg(-1) per day) over a 7-day period in female SD rats using metabonomic analyses of (1) H NMR spectra of urine, serum and liver tissue extracts. Histopathological studies of liver and analyses of blood biochemical parameter, such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen and creatinine revealed that CX had no negative impacts on liver and kidney. However, the metabolic signature of (1) H NMR-based urinalysis of daily samples displayed an increment in the levels of taurine, trimethylamine-N-oxide (TMAO), betaine and acetate. Elevated serum levels of creatinine, glucose, alanine, TMAO and betaine and lower levels of lactate were observed. Metabolic profiling on aqueous soluble extracts of liver showed simultaneously increases in succinate, glycogen, choline, glycerophosphorylcholine, TMAO and betaine levels and reduction in valine, glucose and lactate levels. Nevertheless, no changes in any metabonomic level were found in lipid-soluble extracts of liver. These findings indicate that CX has a slight toxicity in liver and kidney via disturbance of the metabolisms of energy and amino acids. The present study provides a reasonable explanation for the clinical hepatotoxicity of Cimicifuga extract.

Mass spectrometry based metabolomics to study the development of Non-alcoholic steatohepatitis

It can be hypothesized that the development of non-alcoholic steatohepatitis (NASH) is accompanied by severe changes at the metabolic level. Studying the metabolome may reveal insights in the underlying mechanisms leading from steatosis to the development of NASH and its progression to cirrhosis. Metabolomics attempts the comprehensive and quantitative analysis of metabolites in a biological system. Two complementary approaches are used for metabolomic investigations: metabolic fingerprinting and metabolic profiling. Metabolic fingerprinting aims to identify patterns or “fingerprints“ of metabolites, which change in response to perturbations. In metabolic profiling, quantitative analytical methods are developed for metabolites in a metabolic pathway.This contribution will present two robust analytical methods for metabolic profiling and metabolic fingerprinting that were used to quantitatively probe changes in the metabolome associated with the development of NASH. Metabolic fingerprinting was performed by gas chromatography coupled to mass spectrometry (GC-MS). Furthermore, an LC–ESI-MS/MS method was developed for the quantitative determination of 14 intermediates of the methionine and polyamine pathways with lower limits of quantification in the range of 5.0–500 nM [1]. Preliminary studies were performed with samples from a long-term feeding study. BALB/c mice were fed a control or a high fat diet. Liver tissue extracts were subjected to metabolic fingerprinting by GC-MS. A clear segregation of the two groups was observed in a principal component analysis. The identification of discriminating metabolites is in progress. Metabolic changes in the methionine and polyamine pathways were analyzed in methanolic liver extracts. The preliminary data from the liver extracts of mice fed the paigen diet show a significant increase for purescine and 5-deoxy-5-(methylthio)adenosine, while S-adenosyl -homocysteine, spermidine, spermine and acetylspermine were down regulated.

Autoimmune processes after long-term low-level exposure to electromagnetic fields (experimental results) part 3. The effect of long-term nonthermal RF EMF exposure on complement-fixation antibodies against homologenous tissue

The effect of long-term nonthermal EMF exposure of 2450 MHz (500 μW/cm2) on the level of antibodies against brain and liver tissues is studied on rats by the complement-fixation test (CFT) in the cold. It is found that a statistically significant increase in the level of antibodies against aqueous extracts of brain and liver tissues in blood serum is detected only on the 14th day after exposure is ended.

Chymase inhibitor prevents the nonalcoholic steatohepatitis in hamsters fed a methionine‐ and choline‐deficient diet

Mast cells may be involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). The mast cell protease chymase contributes to the formation of angiotensin II and matrix metalloproteinase (MMP)-9, both of which are intimately involved in liver fibrosis. Therefore, we hypothesized that chymase plays an important role in the development of NASH. Hamsters were fed a methionine- and choline-deficient (MCD) diet for 8 weeks. These animals were divided into two groups and received either TY-51469 (1 mg/kg per day) or placebo. A third group was fed a normal diet as a control. Total plasma bilirubin, triglycerides, and hyaluronic acid levels were significantly higher in the MCD diet-fed hamsters than in the normal diet-fed hamsters, but the levels were significantly lower in chymase inhibitor-treated MCD diet-fed hamsters than in placebo-treated MCD diet-fed hamsters. Using histological analysis, marked steatosis and fibrosis were observed in MCD diet-fed hamsters, but these changes were significantly attenuated by treatment with the chymase inhibitor. Increases in mast cells and chymase-positive cells were observed in the liver after the MCD diet, but the increases disappeared in the chymase inhibitor-treated group. The significant increase observed in chymase activity in liver tissue extract from the MCD diet-fed group was also reduced by treatment with the chymase inhibitor. Chymase inhibition significantly reduced not only angiotensin II expression but also matrix metallopeptidase 9 activity in MCD diet-fed hamsters. These findings demonstrate that the mast cell protease chymase may play a crucial role in the development of NASH in hamsters.

Circadian control of XPA and excision repair of cisplatin-DNA damage by cryptochrome and HERC2 ubiquitin ligase

Cisplatin is one of the most commonly used anticancer drugs. It kills cancer cells by damaging their DNA, and hence cellular DNA repair capacity is an important determinant of its efficacy. Here, we investigated the repair of cisplatin-induced DNA damage in mouse liver and testis tissue extracts prepared at regular intervals over the course of a day. We find that the XPA protein, which plays an essential role in repair of cisplatin damage by nucleotide excision repair, exhibits circadian oscillation in the liver but not in testis. Consequently, removal of cisplatin adducts in liver extracts, but not in testis extracts, exhibits a circadian pattern with zenith at approximately 5 pm and nadir at approximately 5 am. Furthermore, we find that the circadian oscillation of XPA is achieved both by regulation of transcription by the core circadian clock proteins including cryptochrome and by regulation at the posttranslational level by the HERC2 ubiquitin ligase. These findings may be used as a guide for timing of cisplatin chemotherapy.

Laser desorption/ionization mass spectrometry on nanostructured semiconductor substrates: DIOS™ and QuickMass™

In the era of systems biology, new analytical platforms are under demand. Desorption/ionization on silicon mass spectrometry (DIOS-MS) is a promising high throughput laser mass spectrometry approach that has attracted a lot of attention, and has been commercialized. Another substrate material manufactured by physical method has also been made commercially available under the trade name of QuickMass™. These two commercial substrates, DIOS™ and QuickMass™, were investigated independently from the manufacturers and were characterized by a number of advanced surface techniques. This work determined (1) the correlation between the substrate physicochemical properties and their LDI activity, (2) the feasibility of metabolic profiling from complex biological matrices and (3) the laser desorption/ionization mechanism. The DIOS™ substrate was characterized with a thick nano-sized porous layer, a high surface concentration of fluorocarbon and silicon oxides and super-hydrophobicity. In contrast, the QuickMass™ substrate consisted of a non-porous germanium thin-film. The relatively high ionization efficiency obtained from the DIOS™ substrate was contributed to the fluorosilane manufacturing processes and its porous morphology. Despite the QuickMass™ substrate being less effective, it was noted that the use of germanium affords a self-cleaning mechanism and suppresses background interference of mass spectra. The suitability of DIOS™ substrates for metabolic profiling of complex biological matrices was demonstrated. DIOS mass spectra of human blood plasma, human urine and animal liver tissue extracts were produced. Suitable extraction methods were found to be important, but relatively simplified approaches were sufficient. Further investigations of the DIOS desorption/ionization mechanism were carried out. The previously proposed sub-surface state reaction could be a molten-solid interfacial state reaction of the substrate and this had a significant effect toward the protonation reaction of amines.

Metabolomics investigation of exercise-modulated changes in metabolism in rat liver after exhaustive and endurance exercises

Exhaustive exercise and endurance exercise training modify the physiological status of the body differently. The present study aimed to evaluate the alteration in biochemical composition with exhaustive and endurance exercises in rats using metabolomics strategy. The metabolite profile of liver tissue was investigated on gas chromatography-mass spectrometry (GC-MS). Data further underwent partial least-squares-discriminant analysis (PLS-DA) to compare the effects on metabolites in sedentary control, exhaustively exercised and endurance trained rats. GC-MS detected 115 highly reproducible peaks in chromatograms from individual liver tissue extracts, and we identified 55 of them. The three groups showed significant differences in metabolic profile. Changes in liver metabolism involved metabolites such as amino acids, fatty acids, organic acids, and carbohydrates. Endurance training elevated the greater rate of tricarboxylic acid cycle and antioxidant activity, and exhaustive exercise led to accumulated urea markers and an inflammation response in liver. In addition, GC-MS-based metabolomic analysis is a promising tool to investigate a pathological status with different exercise programs.

Improved insulin sensitivity by calorie restriction is associated with reduction of ERK and p70S6K activities in the liver of obese Zucker rats

Calorie restriction (CR) improves obesity-related insulin resistance through undefined molecular mechanisms. Insulin receptor substrate (IRS)-1 serine/threonine kinases have been proposed to modulate insulin sensitivity through phosphorylation of IRS proteins. The aim of this study is to test the hypothesis that changes in the activity of IRS1 serine/threonine kinases may underlie the molecular mechanism of CR in improving insulin sensitivity. Obese and lean Zucker rats were subjected to 40% CR or allowed to feed ad libitum (AL) for 20 weeks; body weight and insulin sensitivity were monitored throughout this period. The activity of IRS1 serine/threonine kinases - including JNK, ERK, MTOR/p70(S6K) (RPS6KB1 as listed in the MGI Database), glycogen synthase kinase 3beta (GSK3B), AMPK (PRKAA1 as listed in the MGI Database), and protein kinase C (PRKCQ) in liver tissue extracts was measured by an in vitro kinase assay using various glutathione-S-transferase (GST)-IRS1 fragments as substrates, while phosphorylation of IRS1 and serine kinases was determined by western blotting using phosphospecific antibodies. CR in obese rats significantly reduced body weight and increased insulin sensitivity compared to AL controls. Serine kinase activity toward IRS1(S612) (corresponding to S616 in human IRS1) and IRS1(S632/635) (corresponding to S636/639 in human IRS1) was increased in obese rats compared to lean littermates, and was markedly decreased following CR. Concomitantly, obesity increased and CR decreased the activity of hepatic ERK and p70(S6K) against IRS1. The close association between the activity of hepatic ERK and p70(S6K) with insulin resistance suggests an important role for ERK and p70(S6K) in the development of insulin resistance, presumably via phosphorylation of IRS proteins.

Misregulation of the arginase pathway in tissues of spontaneously hypertensive rats

There is a growing evidence that arginase has a role in the pathophysiology of cardiovascular diseases including hypertension. We recently reported arginase upregulation in aortas from hypertensive spontaneously hypertensive rats (SHRs). The aim of this study was to determine whether arginase abnormalities occur in other tissues of SHR, including the target organs of hypertension. Experiments were conducted on 5-, 10-, 19- and 26-week-old SHRs and Wistar-Kyoto (WKY) rats. Arginase activity and expression were evaluated in heart, kidney, liver, lung and brain tissue extracts. To investigate the role of blood pressure by itself in arginase abnormalities, arginase activity was determined in 10-week-old SHRs previously treated with hydralazine (20 mg kg(-1) per day, for 5 weeks). Compared with WKY rats, cardiac arginase activity was higher in hypertensive SHRs aged 10 weeks (+46%, P<0.05), 19 weeks (+29%, P<0.05) and 26 weeks (+23%, NS). Similar results were found in lungs in which arginase activity was increased in SHRs aged 10 weeks (+39%, P<0.05), 19 weeks (+49%, P<0.05) and 26 weeks (+36%, P<0.05) compared with WKY rats. The changes in arginase activity in these tissues were not associated with changes in enzyme expression. The prevention of hypertension by hydralazine blunted the increase in arginase activity in the hearts but not in the lungs. No change in arginase activity/expression was found in the kidney, liver or brain. In conclusion, this study shows that increased arginase activity is not restricted to large vessels in SHRs and suggests that cardiac arginase activity is hemodynamic sensitive.

Evaluation of Endocrine Disruptor Levels in Kafue Lechwe (kobus leche kafuensis) Samples from the Blue Lagoon National Park of Zambia

The concentrations of endocrine disruptors were determined in 36 liver tissue, serum and whole blood sample extracts drawn from 15 Blue Lagoon National Park Kafue lechwe. Out of 10 analytes evaluated, 89% of the sample extracts showed very high dieldrin concentrations of between 0.08–100 μg/mL in serum, 0.08–24.8 μg/mL in whole blood and 0.08–4.6 μg/g wet weight in liver tissue extracts. pp-DDE was detected in 83% of the sample extracts at 0.006–5.1 μg/mL in serum, 0.006–8.5 μg/mL in whole blood and 0.006–0.12 μg/g wet weight in liver tissue extracts. There was strong correlation between pp-DDE and dieldrin in all the three matrices. Deltamethrin and endosulfan detected at 50% frequency each. Percent recoveries in spiked laboratory blanks ranged between 60–100% while calculated detection limits ranged from 0.004 to 0.21 μg/mL for all the endocrine disruptors evaluated.

AMP-activated protein kinase and carbohydrate response element binding protein: A study of two potential regulatory factors in the hepatic lipogenic program of broiler chickens

This study investigated the effects of fasting and refeeding on AMP-activated protein kinase (AMPK) and carbohydrate response element binding protein (ChREBP) mRNA, protein and activity levels; as well as the expression of lipogenic genes involved in regulating lipid synthesis in broiler chicken (Gallus gallus) liver. Fasting for 24 or 48 h produced significant declines in plasma glucose (at 24 h), insulin and thyroid hormone (T3) levels that were accompanied by changes in mRNA expression levels of hepatic lipogenic genes. The mRNA levels of malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase alpha (ACCalpha), fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1) and thyroid hormone responsive Spot 14 (Spot 14) declined in response to fasting. Refeeding for 24 h increased mRNA levels for each of these genes, characterized by a significant increase ('overshoot') above fed control values. No change in mRNA expression of the two AMPK alpha subunit genes was observed in response to fasting or refeeding. In contrast, ChREBP and sterol regulatory element binding protein-1 (SREBP-1) mRNA levels decreased during fasting and increased with refeeding. Phosphorylation of AMPK alpha subunits increased modestly after a 48 h fast. However, there was no corresponding change in the phosphorylation of ACC, a major downstream target of AMPK. Protein level and DNA-binding activity of ChREBP increased during fasting and declined upon refeeding as measured in whole liver tissue extracts. In general, evidence was found for coordinate transcriptional regulation of lipogenic program genes in broiler chicken liver, but specific regulatory roles for AMPK and ChREBP in that process remain to be further characterized.

In vivo MRS assessment of altered fatty acyl unsaturation in liver tumor formation of a TGFα/c-myc transgenic mouse model

Current detection methods (computed tomography, ultrasound, and MRI) for hepatocarcinogenesis in humans rely on visual confirmation of neoplastic formations. A more effective early detection method is needed. Using in vivo magnetic resonance spectroscopy (MRS), we show that alterations in the integral ratios of the bis-allyl to vinyl hydrogen protons in unsaturated lipid fatty acyl groups correlate with the development of neoplastic formations in vivo in a TGFalpha/c-myc mouse hepatocellular carcinoma (HCC) model. HPLC analysis of the TGFalpha/c-myc mice liver tissue revealed a significant increase in the amount of oleic acid, along with alterations in linoleic and gamma-linolenic acids, as compared with control CD1 mice. Electrospray ionization tandem mass spectrometry analysis indicated a significant increase in the abundance of specific glycerol phosphatidylcholine (GPCho) lipids containing palmitic and oleic acids between control CD1 and TGFalpha/c-myc mice liver tissue extracts. Western blot analysis of the mice liver tissue indicates alterations in the desaturase enzyme stearoyl CoA desaturase (SCD)1, responsible for palmitic and oleic acid formation. Microarray analysis detected alterations in several genes involved with fatty acid metabolism, particularly SCD2, in transgenic mouse liver tissue. In correlation with the HPLC, mass spectrometry, Western blot, and microarray analyses, we are able to confirm the ability of in vivo MRS to detect precancerous lesions in the mouse liver before visual neoplastic formations were detectable by MRI.

A multi-analytical approach for metabolomic profiling of zebrafish (Danio rerio) livers

A metabolomic study was performed to investigate the biochemical profiles of livers from male and female zebrafish (Danio rerio), using a multiple platform approach, incorporating 1H NMR, GC/MS and LC/MS. The reproducibility and reliability of the three methods were validated prior to the assays. Major biomolecules detected using one method were also cross examined using the other techniques. These metabolites included carbohydrates, lipids, amino acids detected using 1H NMR and GC/MS, and acetylcarnitine, choline and various phospholipids determined using 1H NMR and LC/MS. Our findings suggest that 1H NMR provided comprehensive information on glucose, amino acids, pyruvate and other smaller biochemical constituents of the zebrafish liver. On the other hand, GC/MS was able to assay cholesterol, saturated and unsaturated fatty acids, and LC/MS was ideal for the analysis of lipids/phospholipids. These techniques revealed that there are significant differences in the biochemical profiles of male and female zebrafish liver tissue extracts. Specifically, we noted that although there were no significant differences observed for the carbohydrate profile, the amino acid profile was rather different in male and female zebrafish liver. Furthermore, data from all three techniques revealed that although the saturated fatty acid profile was similar, the compositions of unsaturated fatty acids were different in the two phenotypes. The overall findings suggested that this multiplatform approach offers comprehensive coverage of a metabolome as well as provides valuable insight towards understanding the different biochemical profiles of a biosystem.

Transcriptomic and Metabonomic Profiling of Obesity-Prone and Obesity-Resistant Rats under High Fat Diet

Rodents respond to chronic high fat diet in at least two ways: some of them may readily gain body weight and become obese (termed obesity-prone, OP), and others may not (termed obesity-resistant, OR). Transcriptomic and metabonomic profiling of OP and OR rats has been conducted, showing two sets of significantly different phenotypic profiles in response to 16 weeks of high fat diet. We observed significant differences in transcriptional expression of nearly 80 genes, some of which are known to be involved in lipid metabolism, transport, and ketone body production. The different metabolic profiles in liver tissue extracts, serum, and urine between the two phenotypes can be ascribed to the corresponding pathways identified with multivariate statistical analysis, including fatty acid metabolism, Krebs cycle, and amino acid metabolism. The integration of results from transcriptomic and metabonomic studies revealed that the altered metabolic pathways in OP rats may involve the increased activity of sympathetic nervous system and Krebs cycle, an increased production of ketone bodies, and an adaptive regulatory process to store excessive lipids in liver through reverse cholesterol transport process. These biochemical variations at transcriptional and metabolic levels as a result of dietary intervention highlight the significance of combined "omics" strategy in the mechanistic study of obesity and metabolic disorders.

Hematological Toxicity Associated with Tissue Extract from Poisonous Fish Lagocephalus lagocephalus—Influence on Erythrocyte Function in Wistar Rats

The puffer fish Lagocephalus lagocephalus represents serious public health problems in the world. The relative toxicity of each organ (liver and flesh) was determined by the relation dose-death time "mouse bioassay." The average liver toxicity of the puffer fish was the highest when compared with flesh giving 14.32 and 10.88 MU/g, respectively. A mouse unit is the amount of toxin (extract of fish organ) that kills a 20 g male mouse in 30 min after intraperitoneal injection. One mouse unit is equivalent to 0.22 microg of TTX. For the rat bioassay tests, Wistar rats were daily i.p. injected, for 10 d, with extracts of liver (LT) or flesh (FT) (muscles + skin) of L. lagocephalus. Control rats received injection of NaCl (0.9%). During the experiment, a significant reduction in red blood cell number (RBC), hemoglobin (HGB) concentration, and hematocrit (HCT) was observed essentially after 10 d of treatment in the FT and LT-exposed groups. Consequently, treatment led to severe anemia and hemolytic action as indicated by a significant reduction in the total number of erythrocytes. In fact, our study revealed a significant increase in erythrocyte lipid peroxidation (LPO) in FT and LT groups compared with controls after experimental exposure. The flesh and liver tissue extracts also altered antioxidative enzymes activities: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Histopathological alterations in the spleen occurred exclusively at the end of treatment. We marked also an increase in reticulo-endothelial cells, which led to remove damaged erythrocytes.

Integrated metabolomic analysis of the nano-sized copper particle-induced hepatotoxicity and nephrotoxicity in rats: A rapid invivo screening method for nanotoxicity

Despite an increasing application of copper nanoparticles, there is a serious lack of information concerning their impact on human health and the environment. In this study, the biochemical compositions of urine, serum, and extracts of liver and kidney tissues of rats treated with nano-copper at the different doses (50, 100, and 200 mg/kg/d for 5 d) were investigated using 1H NMR techniques with the pattern recognition methods. Serum biochemical analysis and histopathological examinations of the liver and kidney of all the rats were simultaneously performed. All the results indicated that the effects produced by nano-copper at a dose of 100 or 50 mg/kg/d were less than those induced at a higher dose of 200 mg/kg/d. Nano-copper induced overt hepatotoxicity and nephrotoxicity at 200 mg/kg/d for 5 d, which mainly involved scattered dot hepatocytic necrosis and widespread renal proximal tubule necrosis. Increased citrate, succinate, trimethylamine- N-oxide, glucose, and amino acids, accompanied by decreased creatinine levels were observed in the urine; furthermore, elevated levels of lactate, 3-hydroxybutyrate, acetate, creatine, triglycerides, and phosphatide and reduced glucose levels were observed in the serum. The predominant changes identified in the liver tissue aqueous extracts included increased lactate and creatine levels together with reduced glutamine and taurine levels, and the metabolic profile of the kidney tissue aqueous extracts showed an increase in lactate and a drop in glucose. In the chloroform/methanol extracts of the liver and kidney tissues, elevated triglyceride species were identified. These changes suggested that mitochondrial failure, enhanced ketogenesis, fatty acid β-oxidation, and glycolysis contributed to the hepatotoxicity and nephrotoxicity induced by nano-copper at 200 mg/kg/d for 5 d. An increase in triglycerides in the serum, liver and kidney tissues could serve as a potential sensitive biomarker reflecting the lipidosis induced by nano-copper. The data generated from the current study completely supports the fact that an integrated metabolomic approach is promising for the development of a rapid in vivo screening method for nanotoxicity.

Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish

The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4β-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4α-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish.

Rapid and sensitive HPLC assay for simultaneous determination of procaine and para-aminobenzoic acid from human and rat liver tissue extracts

A sensitive and rapid high-performance liquid chromatography method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA) from human and rat liver tissue extracts. The method has been validated according to ICH guidelines in terms of selectivity, linearity, lower limit of detection, lower limit of quantitation, accuracy, precision and recovery from human and rat liver tissue extracts. Chromatography was carried out on a Discovery ® C 18 column using 10 mM ammonium acetate at pH 4.0 and acetonitrile as mobile phase. Retention times for procaine and PABA were 6.6 and 5.3 min, respectively. Linearity for each calibration curve in both tissue extracts was observed across a range from 10 μM to 750 μM for procaine and PABA. The lower limit of detection for both procaine and PABA was 5 μM and the lower limit of quantitation was 10 μM in both tissue extracts. The intra- and inter-day relative standard deviations (R.S.D.) for both procaine and PABA were <6%. Recoveries of procaine and PABA from human and rat liver tissue extracts were determined by two different methods with a single-step protein precipitation technique being employed in both methods. Recoveries for both procaine and PABA were greater than 80% from both human and rat liver tissue extracts.