Safety assessment of enzymatically converted chicken bile as a novel food material: Genotoxicity, teratogenicity, and acute and subchronic toxicity studies.

Enzymatically converted chicken bile (CB), prepared by converting taurine deoxycholic acid (TCDCA) to taurine ursodeoxycholic acid (TUDCA) in CB, possesses various functional activities. But their nutrient composition and safety assessment have not been fully investigated yet. CB was mainly composed of proteins and steroids. CB did not show genotoxic effects based on Ames test, mammalian erythrocyte micronucleus test, and in vitro mammalian chromosomal aberration test. There were no growth abnormalities or deaths in the acute toxicity test for mice, indicating that CB is nontoxic with an LD50 > 10 g/kg·body weight (BW). Subchronic toxicity test and genotoxicity test were performed based on intake of 0.5 g CB per person daily at expanded doses of 33.3, 100, and 300 times (278, 833, and 2500 mg/kg·BW). The result indicated that CB at 833 mg/kg·BW showed no toxicity on BW, body weight gain, food intake, hematological, serum biochemistry, absolute/relative organ weights, urinalysis, and pathological features of rats in the subchronic toxicity test, while CB at 833 mg/kg·BW induced maternal toxicity with no fetus teratogenicity or embryotoxicity in the teratogenicity test. In conclusion, CB did not show toxic effects and a long-term daily intake of CB at 0.5 g per person is considered safe, but pregnant women should avoid it. These findings could provide a reference for the safe use of CB in functional food.

Viability assessment of the liver during ex-situ machine perfusion prior to transplantation.

PURPOSE OF REVIEW: In an attempt to reduce waiting list mortality in liver transplantation, less-than-ideal quality donor livers from extended criteria donors are increasingly accepted. Predicting the outcome of these organs remains a challenge. Machine perfusion provides the unique possibility to assess donor liver viability pretransplantation and predict postreperfusion organ function. RECENT FINDINGS: Assessing liver viability during hypothermic machine perfusion remains challenging, as the liver is not metabolically active. Nevertheless, the levels of flavin mononucleotide, transaminases, lactate dehydrogenase, glucose and pH in the perfusate have proven to be predictors of liver viability. During normothermic machine perfusion, the liver is metabolically active and in addition to the perfusate levels of pH, transaminases, glucose and lactate, the production of bile is a crucial criterion for hepatocyte viability. Cholangiocyte viability can be determined by analyzing bile composition. The differences between perfusate and bile levels of pH, bicarbonate and glucose are good predictors of freedom from ischemic cholangiopathy. SUMMARY: Although consensus is lacking regarding precise cut-off values during machine perfusion, there is general consensus on the importance of evaluating both hepatocyte and cholangiocyte compartments. The challenge is to reach consensus for increased organ utilization, while at the same time pushing the boundaries by expanding the possibilities for viability testing.

Sampling time for different matrices in stress assessment of farmed Atlantic salmon post-smolt.

The sustainability of commercial aquaculture production depends critically on prioritizing fish welfare management. Besides monitoring welfare parameters such as fish behaviour and water quality, fish stress level can also provide a reliable measure of the welfare status of farmed fish. Cortisol and 5 of its metabolites (5ß-THF, cortisone, 5ß-DHE, 5ß-THE, ß-cortolone) were previously identified by the authors as suitable stress biomarkers of farmed Atlantic salmon. Based on this knowledge, the present study aimed to investigate the time-related dynamics of these metabolites in plasma, skin mucus, bile and faeces over a 72 h- period. The objective was to determine the optimal sampling time for each matrix and to understand the clearance pathway of these metabolites following stress. An experiment was carried out using a total of 90 Atlantic salmon with an average weight of 438 (±132) g. The average sea temperature was 6.9 °C during the experimental period. A control group of 10 fish was first collected before the remaining 80 fish were submitted to a stress of netting and subsequent relocation into two separate cages. From each of these two stress groups, 10 fish were sampled at 1 h, 2 h, 4 h, 6 h and 12 h, 24 h, 48 h, 72 h after the stress event respectively. The concentrations of cortisol and its metabolites were measured at each of the sampling timepoint. The results demonstrated that plasma cortisol metabolites reached the highest concentration 4 h after stress and remained elevated despite the slight decrease for the remaining timepoints. The peak level was observed at 12 h post-stress in skin mucus and 24 h in bile and faeces. The findings suggest that these timepoints are the optimal for sampling Atlantic salmon post-smolt following stressful events in acute stress studies. Furthermore, the results reveal that analysing cortisol and its metabolites, both in free and conjugated forms, rather than free cortisol provides greater flexibility as their concentrations are less affected by sampling procedure. This study confirms the appropriateness of skin mucus and faeces as less-invasive sample matrices for fish stress evaluation and provides a basis for further developing low invasive tools for monitoring the welfare of farmed salmonid.

Influence of cholecystectomy on the flow dynamic pattern of bile in the extrahepatic bile duct: Assessment by cine-dynamic MRCP with spatially-selective IR pulse.

PURPOSE: To evaluate the influence of cholecystectomy on the flow dynamic pattern of bile in the extrahepatic bile duct by using cine-dynamic MRCP with spatially-selective inversion-recovery (IR) pulse non-invasively. MATERIALS AND METHODS: 56 patients with cholecystectomy and 48 control subjects without cholecystectomy who underwent cine-dynamic MRCP with spatially-selective IR pulse at 1.5 T or 3 T (TR/TE, 4000 msec/500 msec; echo train spacing, 6.5 msec; echo train length, 172; section thickness, 50 mm; matrix, 320 × 320; field of view, 320 × 320 mm; bandwidth, 488 Hz; and inversion time, 2200 msec). In cine-dynamic MRCP, IR pulse with 20 mm width was placed on the common bile duct (CBD) to evaluate the movement of bile (antegrade and reversed bile flow). Cine-dynamic MRCP imaging was scanned every 15 s (imaging, 4 s; rest, 11 s) during 5 min to acquire a series of single-shot images (a total of 20 images). The frequency that antegrade or reversed bile flow was observed in the extrahepatic bile duct, and 5-point grading score based on the moving distance of antegrade or reversed bile flow were compared between the groups. Both groups were compared using the χ2 and Mann-Whitney U tests (P < 0.05 considered significant). RESULTS: Antegrade bile flow was observed more frequently in the cholecystectomy group than in the non-cholecystectomy group (5.1 times vs. 2.8 times, P = 0.008). Mean grading score of antegrade bile flow was significantly greater in the cholecystectomy group than in the non-cholecystectomy group (mean grade, 0.33 vs 0.21; P = 0.014). Regarding reversed bile flow, there were no significant differences in the frequency and grading score between cholecystectomy group and non-cholecystectomy group. CONCLUSION: Antegrade bile flow was observed more frequently and predominantly in patients after cholecystectomy in cine-dynamic MRCP with spatially-selective IR pulse while reversed bile flow was observed equivalently.

Lipid metabolism and functional assessment of discarded human livers with steatosis undergoing 24 hours of normothermic machine perfusion.

Normothermic machine perfusion (NMP) is an emerging technology to preserve liver allografts more effectively than cold storage (CS). However, little is known about the effect of NMP on steatosis and the markers indicative of hepatic quality during NMP. To address these points, we perfused 10 discarded human livers with oxygenated NMP for 24 hours after 4-6 hours of CS. All livers had a variable degree of steatosis at baseline. The perfusate consisted of packed red blood cells and fresh frozen plasma. Perfusate analysis showed an increase in triglyceride levels from the 1st hour (median, 127 mg/dL; interquartile range [IQR], 95-149 mg/dL) to 24th hour of perfusion (median, 203 mg/dL; IQR, 171-304 mg/dL; P = 0.004), but tissue steatosis did not decrease. Five livers produced a significant amount of bile (≥5 mL/hour) consistently throughout 24 hours of NMP. Lactate in the perfusate cleared to <3 mmol/L in most livers within 4-8 hours of NMP, which was independent of bile production rate. This is the first study to characterize the lipid profile and functional assessment of discarded human livers at 24 hours of NMP. Liver Transplantation 24 233-245 2018 AASLD.

Analysis of the Change in the Blood Concentration-Time Profile Caused by Complex Drug-Drug Interactions in the Liver Considering the Enterohepatic Circulation: Examining Whether the Inhibition Constants for Uptake, Metabolism, and Biliary Excretion Can be Recovered by the Analyses Using Physiologically Based Pharmacokinetic Modeling.

Hypothetical substrates undergoing transporter-mediated hepatic uptake, metabolism, and enterohepatic circulation with different rate-determining processes with a combination of inhibition constants (Ki) for hepatic uptake, metabolism, and biliary excretion processes were generated with a constant Ki for uptake and incorporated into a physiologically based pharmacokinetic model. Analyses of the kinetic model suggested that the fraction of substrates excreted in the bile to the total elimination by the liver (fbile) can be estimated under certain conditions from kinetic analyses of their blood concentration-time profiles. Using the generated time profiles of substrates with and without coadministration of inhibitors, various pharmacokinetic parameters involving fbile and Ki for the hepatic uptake, metabolism, and biliary excretion of drugs were back-calculated by fitting. Comparing parameters obtained with the original parameter sets by fitting, the Ki were found to be well estimated under the following conditions: the initial estimates for inhibition constants were relatively good, which corresponds to the case for obtaining reliable in vitro inhibition constants. In conclusion, the integration of top-down analyses with bottom-up estimates (experimental determination) of inhibition constants can be used to estimate in vivo inhibition constants and fbile reliably.

An accelerated background subtraction algorithm for processing high-resolution MS data and its application to metabolite identification.

BACKGROUND: Metabolite identification without radiolabeled compound is often challenging because of interference of matrix-related components. RESULTS: A novel and an effective background subtraction algorithm (A-BgS) has been developed to process high-resolution mass spectral data that can selectively remove matrix-related components. The use of a graphics processing unit with a multicore central processing unit enhanced processing speed several 1000-fold compared with a single central processing unit. A-BgS algorithm effectively removes background peaks from the mass spectra of biological matrices as demonstrated by the identification of metabolites of delavirdine and metoclopramide. CONCLUSION: The A-BgS algorithm is fast, user friendly and provides reliable removal of matrix-related ions from biological samples, and thus can be very helpful in detection and identification of in vivo and in vitro metabolites.

Morphological and Functional Characterization and Assessment of iPSC-Derived Hepatocytes for In Vitro Toxicity Testing.

Drug-induced liver injury (DILI) remains a great challenge and a major concern during late-stage drug development. Induced pluripotent stem cells (iPSC) represent an exciting alternative in vitro model system to explore the role of genetic diversity in DILI, especially when derived from patients who have experienced drug-induced hepatotoxicity. The development and validation of the iPSC-derived hepatocytes as an in vitro cell-based model of DILI is an essential first step in creating more predictive tools for understanding patient-specific hepatotoxic responses to drug treatment. In this study, we performed extensive morphological and functional analyses on iPSC-derived hepatocytes from a commercial source. iPSC-derived hepatocytes exhibit many of the key morphological and functional features of primary hepatocytes, including membrane polarity and production of glycogen, lipids, and key hepatic proteins, such as albumin, asialoglycoprotein receptor and α1-antitrypsin. They maintain functional activity for many drug-metabolizing enzyme pathways and possess active efflux capacity of marker substrates into bile canalicular compartments. Whole genome-wide array analysis of multiple batches of iPSC-derived cells showed that their transcriptional profiles are more similar to those from neonatal and adult hepatocytes than those from fetal liver. Results from experiments using prototype DILI compounds, such as acetaminophen and trovafloxacin, indicate that these cells are able to reproduce key characteristic metabolic and adaptive responses attributed to the drug-induced hepatotoxic effects in vivo. Overall, this novel system represents a promising new tool for understanding the underlying mechanisms of idiosyncratic DILI and for screening new compounds for DILI-related liabilities.

Criteria for viability assessment of discarded human donor livers during ex vivo normothermic machine perfusion.

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37 °C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥ 30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial.

Assessment of bile and serum mucin5AC in cholangiocarcinoma: diagnostic performance and biologic significance.

BACKGROUND: Recent studies have showed the efficacy of mucin5AC (MUC5AC) as a diagnostic and prognostic serum biomarker in biliary tract tumors. The aim of the present investigation was to improve the current knowledge on the biologic relevance of MUC5AC in malignant and benign biliary disorders by comparing its diagnostic performance in both bile and serum samples of patients with cholangiocarcinoma (CCA) or benign biliary disorders. METHODS: A quantitative determination of MUC5AC by enzyme-linked immunosorbent assay was performed in bile and serum specimens from 26 patients with extrahepatic CCA and 20 subjects with benign biliary disorders (10 with biliary stones and 10 with cholangitis). Verification analysis was made by immunoblot. RESULTS: MUC5AC of serum and biliary origin contributed to different extent to total levels of MUC5AC in the different groups of patients. In particular, the transition toward a greater degree of injury of bile duct epithelium was accompanied by a greater amount of MUC5AC in serum than in bile. The diagnostic performance of MUC5AC expressed as serum/bile ratio showed excellent diagnostic performance for differentiating CCA from cholangitis (area under the curve [AUC], 0.94; 95% CI, 0.86-1.00; P < .0001), CCA from biliary stones (AUC, 0.99; 95% CI, 0.98-1.00; P < .0001), as well as cholangitis from biliary stones (AUC, 0.93; 95% CI, 0.82-1.00; P = .001). CONCLUSION: These findings provide new insight into the biologic importance of MUC5AC in biliary disorders and suggest that combined assessment of MUC5AC in bile and serum with expression of data in terms of serum to bile ratio may improve the diagnostic performance of MUC5AC quantification in serum alone.

Metabolism and excretion of 6-chloro-9-(4-methoxy-3,5-dimethylpyridin-2-ylmethyl)-9H-purin-2-ylamine, an HSP90 inhibitor, in rats and dogs and assessment of its metabolic profile in plasma of humans.

6-Chloro-9-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethyl)-9H-purin-2-ylamine (BIIB021), a synthetic HSP90 inhibitor, exhibited promising antitumor activity in preclinical models and was in development for the treatment of breast cancer. The metabolism and excretion of BIIB021 was investigated in rats and dogs after oral administration of [(14)C]BIIB021. The administered radioactive dose was quantitatively recovered in both species, and feces/bile was the major route of excretion. Metabolic profiling revealed that BIIB021 is extensively metabolized primarily via hydroxylation of the methyl group (M7), O-demethylation (M2), and to a lesser extent by glutathione conjugation (M8 and M9). M7 was further metabolized to form the carboxylic acid (M3) and glucuronide conjugate (M4). Human plasma obtained from the phase I study in cancer patients were also analyzed to assess the metabolism of BIIB021 in humans and to ensure that selected animal species were exposed to all human major metabolites. Results suggested that BIIB021 is metabolized via hydroxylation followed by carboxylation and glucuronidation in humans consistent with rat and dog; however, an additional dominant circulating metabolite, hydroxylation at the purine ring (M10), was identified in humans. Preliminary in vitro studies using liver cytosolic fractions indicated that M10 formation is primarily catalyzed by aldehyde oxidase (AO). AO catalytic activity for M10 formation was the highest in the monkey, followed by mouse, human, and rat. The apparent K(m) and V(max) values of M10 formation were 174 ± 8 µM and 14.0 ± 0.3 pmol•min(-1)•mg protein(-1) in human and 132 ± 9 µM and 131 ± 4 pmol•min(-1)•mg protein(-1) in monkey, respectively.

Assessment of potential drug interactions by characterization of human drug metabolism pathways using non-invasive bile sampling.

AIM: Characterization of the biliary disposition of GSK1325756, using a non-invasive bile sampling technique and spectrometric analyses, to inform the major routes of metabolic elimination and to enable an assessment of victim drug interaction risk. METHOD: Sixteen healthy, elderly subjects underwent non-invasive bile capture using a peroral string device (Entero-Test(®)) prior to and following a single oral dose of GSK1325756 (100 mg). The device was swallowed by each subject and once the weighted string was judged to have reached the duodenum, gallbladder contraction was stimulated in order to release bile. The string was then retrieved via the mouth and bile samples were analyzed for drug-related material using spectrometric and spectroscopic techniques following solvent extraction. RESULTS: Nuclear magnetic resonance spectroscopy (NMR) indicated that the O-glucuronide metabolite was the major metabolite of GSK1325756, representing approximately 80% of drug-related material in bile. As bile is the major clearance route for GSK1325756 (only 4% of the administered dose was excreted in human urine), this result indicates that uridine 5'-diphospho-glucuronosyltransferases (UGTs) are the major drug metabolizing enzymes responsible for drug clearance. The relatively minor contribution made by oxidative routes reduces the concern of CYP-mediated victim drug interactions. CONCLUSION: The results from this study demonstrate the utility of deploying the Entero-Test® in early human studies to provide information on the biliary disposition of drugs and their metabolites. This technique can be readily applied in early clinical development studies to provide information on the risk of interactions for drugs that are metabolized and eliminated in bile.

Determination of cnidilin and its two metabolites in rat bile and stool after oral administration by HPLC/electrospray ionization tandem mass spectrometry.

A simple, fast and sensitive method for the simultaneous determination of cnidilin and its two metabolites (M1 and M2) in rat bile and stool using HPLC coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed. The sample pretreatment was simple, because methanol was the only additive used for dilution of bile and ultrasound of stool. Pimpinellin was used as internal standard (IS). The separation was performed on a reverse phase C18 column with gradient elution consisting of 0.5‰ aqueous formic acid and methanol (containing 0.5‰ formic acid). The detection was in the multiple-reaction monitoring mode within 7 min. All the analytes were in accordance with the requirement of the validation of the method in vivo (linearity, precision, accuracy, limit of detection and limit of quantification). After oral administrating 24 mg/kg of the prototype drug cnidilin, M1 and M2 were determined in bile within 36 h, and in stool within 60 h. Cnidilin in bile was completely excreted in 24 h, and the main excretive amount of cnidilin was 80% in the first 6 h, but the drug recovery in bile within 24 h was <1.95%. In stool, the main excretive amount of cnidilin was 95.8% in the first 24 h, and the drug recovery within 48 h was lower than 1.48%.

Assessment of heavy metal and PAH exposure in largemouth bass (Micropterus salmoides) in the Reedy River watershed, South Carolina, USA: a multi-season assessment of metallothionein and bile fluorescence.

Biomarkers can be used as tools to help determine ecological exposure in watershed assessments. In the present study, metallothionein and fixed wavelength bile fluorescence for two-, four-, and five-ring hydrocarbons were used as biomarkers of exposure in largemouth bass (Micropterus salmoides) in the Reedy River watershed located in South Carolina, USA. Fish were sampled from three impoundments and a reference site over three seasons in the same year. Biomarker endpoints were compared to chemical concentrations at each site during each season. Results indicated that despite elevated concentrations of hydrocarbons being present in sediments, the hydrocarbons did not appear to be bioavailable based on bile fluorescence analysis. Bile fluorescence analysis also indicated that the hydrocarbons detected in this watershed were likely of petrogenic origin. Significantly elevated sediment concentrations of Cd, Cr, Cu, Pb, Ni, Ag, Zn, and metallothionein were found in one impoundment, Lake Conestee, compared with the reference site, indicating both the presence and bioavailability of these metals. Seasonal variability of bile fluorescence was limited; however, metallothionein showed elevated concentrations in the spring and summer compared with fall.

Preclinical assessment of the distribution, metabolism, and excretion of S-propargyl-cysteine, a novel H2S donor, in Sprague-Dawley rats.

AIM: To study the distribution, metabolism and excretion of S-propargyl-cysteine (SPRC), a novel hydrogen sulfide (H2S) donor, after oral administration in rats. METHODS: Adult Sprague-Dawley rats were used. The tissue distribution of [(35)S] SPRC-derived radioactivity was measured using a liquid scintillation counter. The plasma protein binding of SPRC was examined using 96-well equilibrium dialysis. The excretion of SPRC in urine, bile and feces was analyzed using the LC-MS/MS method. The major metabolites in rat biomatrices were identified using MRM information-dependent, acquisition-enhanced product ion (MRM-IDA-EPI) scans on API 4000QTrap system. RESULTS: After oral administration of [(35)S]-SPRC at a dose of 75 mg/kg, [(35)S] SPRC-derived radioactivity displayed broad biological distribution in various tissues of rats, including its target organs (heart and brain) with the highest in kidney. On the other hand, the binding of SPRC to human, rat and dog plasma protein was low. Only 2.18% ± 0.61% and 0.77% ± 0.61% of the total SPRC administered was excreted unchanged in the bile and urine. However, neither intact SPRC nor its metabolites were detected in rat feces. The major metabolic pathway in vivo (rat bile, urine, and plasma) was N-acetylation. CONCLUSION: The preliminary results suggest that SPRC possesses acceptable pharmacokinetic properties in rats.

Progressive familial intrahepatic cholestasis and benign recurrent intrahepatic cholestasis: a review.

Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are two rare autosomal recessive disorders, characterized by cholestasis. They are related to mutations in hepatocellular transport system genes involved in bile formation. The differentiation between PFIC and BRIC is based on phenotypic presentation: PFIC is a progressive disease, with evolution to end-stage liver disease. BRIC is characterized by intermittent recurrent cholestatic episodes, with irresistible pruritus, mostly without evident liver damage. Between symptomatic periods, patients are completely asymptomatic. In this article, a short overview of the aetiology, the clinical and diagnostic characteristics and the therapy of both PFIC and BRIC are given.

Toxicity assessment of individual ingredients of synthetic-based drilling muds (SBMs).

Synthetic-based drilling muds (SBMs) offer excellent technical characteristics while providing improved environmental performance over other drilling muds. The low acute toxicity and high biodegradability of SBMs suggest their discharge at sea would cause minimal impacts on marine ecosystems, however, chronic toxicity testing has demonstrated adverse effects of SBMs on fish health. Sparse environmental monitoring data indicate effects of SBMs on bottom invertebrates. However, no environmental toxicity assessment has been performed on fish attracted to the cutting piles. SBM formulations are mostly composed of synthetic base oils, weighting agents, and drilling additives such as emulsifiers, fluid loss agents, wetting agents, and brine. The present study aimed to evaluate the impact of exposure to individual ingredients of SBMs on fish health. To do so, a suite of biomarkers [ethoxyresorufin-O-deethylase (EROD) activity, biliary metabolites, sorbitol dehydrogenase (SDH) activity, DNA damage, and heat shock protein] have been measured in pink snapper (Pagrus auratus) exposed for 21 days to individual ingredients of SBMs. The primary emulsifier (Emul S50) followed by the fluid loss agent (LSL 50) caused the strongest biochemical responses in fish. The synthetic base oil (Rheosyn) caused the least response in juvenile fish. The results suggest that the impact of Syndrill 80:20 on fish health might be reduced by replacement of the primary emulsifier Emul S50 with an alternative ingredient of less toxicity to aquatic biota. The research provides a basis for improving the environmental performance of SBMs by reducing the environmental risk of their discharge and providing environmental managers with information regarding the potential toxicity of individual ingredients.

Assessment of hepatotoxicity potential of drug candidate molecules including kinase inhibitors by hepatocyte imaging assay technology and bile flux imaging assay technology.

Kinases are members of a major protein family targeted for drug discovery and development. Given the ubiquitous nature of many kinases as well as the broad range of pathways controlled by these enzymes, early safety assessments of small molecule inhibitors of kinases are crucial in identifying new molecules with sufficient therapeutic window for clinical development. Failure or attrition of drug candidates in late-stage pipelines due to hepatotoxicity is a significant challenge in the drug development field. Herein we provide detailed methods for the hepatocyte imaging assay technology (HIAT) and the bile flux imaging assay technology (BIAT) to evaluate drug-induced liver injury (DILI) potentials for drug candidates. Optimized culturing methods for primary human hepatocytes, both freshly isolated and prequalified cryopreserved cells, are also presented. The applications of these high-content cellular imaging technologies in the evaluation of p38 and Her2 kinase inhibitors are highlighted to illustrate the usefulness of the research methodology in a compound screening as well as mechanistic investigative setting.

Fat digestion and absorption in spice-pretreated rats.

BACKGROUND: A few common spices are known to stimulate secretion of bile with higher amount of bile acids which play a major role in digestion and absorption of dietary lipids. It would be appropriate to verify if these spices enable efficient digestion and absorption during high-fat intake. In this context, dietary ginger (0.05%), piperine (0.02%), capsaicin (0.015%), and curcumin (0.5%) were examined for their influence on bile secretion, digestive enzymes of pancreas and absorption of dietary fat in high-fat (30%) fed Wistar rats for 8 weeks. RESULTS: These spices enhanced the activity of pancreatic lipase, amylase, trypsin and chymotrypsin by 22-57%, 32-51%, 63-81% and 12-38%, respectively. Dietary intake of spices along with high-fat enhanced fat absorption. These dietary spices increased bile secretion with higher bile acid content. Stimulation of lipid mobilisation from adipose tissue was suggested by the decrease in perirenal adipose tissue weight by dietary capsaicin and piperine. This was also accompanied by prevention of the accumulation of triglyceride in liver and serum in high-fat fed rats. Activities of key lipogenic enzymes in liver were reduced which was accompanied by an increased activity of hormone-sensitive lipase. CONCLUSION: Thus, dietary ginger and other spice compounds enhance fat digestion and absorption in high-fat fed situation through enhanced secretion of bile salts and a stimulation of the activity pancreatic lipase. At the same time, the energy expenditure is facilitated by these spices to prevent the accumulation of absorbed fat.

Tracing of the Bile-chemotactic migration of juvenile Clonorchis sinensis in rabbits by PET-CT.

BACKGROUND: Adult Clonorchis sinensis live in the bile duct and cause clonorchiasis. It is known that the C. sinensis metacercariae excyst in the duodenum and migrate up to the bile duct through the common bile duct. However, no direct evidence is available on the in vivo migration of newly excysted C. sinensis juveniles (CsNEJs). Advanced imaging technologies now allow the in vivo migration and localization to be visualized. In the present study, we sought to determine how sensitively CsNEJs respond to bile and how fast they migrate to the intrahepatic bile duct using PET-CT. METHODOLOGY/PRINCIPAL FINDINGS: CsNEJs were radiolabeled with (18)F-fluorodeoxyglucose ((18)F-FDG). Rabbits with a gallbladder contraction response to cholecystokinin-8 (CCK-8) injection were pre-screened using cholescintigraphy. In these rabbits, gallbladders contracted by 50% in volume at an average of 11.5 min post-injection. The four rabbits examined were kept anesthetized and a catheter inserted into the mid duodenum. Gallbladder contraction was stimulated by injecting CCK-8 (20 ng/kg every minute) over the experiment. Anatomical images were acquired by CT initially and dynamic PET was then carried out for 90 min with a 3-min acquisition per frame. Twelve minutes after CCK-8 injection, about 3,000 (18)F-FDG-labeled CsNEJs were inoculated into the mid duodenum through the catheter. Photon signals were detected in the liver 7-9 min after CsNEJs inoculation, and these then increased in the whole liver with stronger intensity in the central area, presenting that the CsNEJs were arriving at the intrahepatic bile ducts. CONCLUSION: In the duodenum, CsNEJs immediately sense bile and migrate quickly with bile-chemotaxis to reach the intrahepatic bile ducts by way of the ampulla of Vater.