Response of broiler breeder pullets when fed hydrolyzed whole yeast from placement to 22 wk of age.

The study examined the effects of feeding broiler breeder pullets hydrolyzed whole yeast (HY) from hatch to 22 wk of age (WOA). A total of 524-day-old Ross 708 pullets were placed in floor pens (∼24 birds/pen) for the starter (0-4 WOA) and grower (5-18 WOA) phases, then transferred to the egg production facility and redistributed to ∼20 birds/pen for the prelay phase (19-22 WOA). Two diets were allocated to pens (0-18 WOA; n = 11) and (19-22 WOA; n-12). The diets were a control corn and soybean meal diet formulated to meet specifications and control plus 0.05% HY (HY). Birds had ad libitum access to feed in the first week and daily feed allocation based on pen BW from 2 WOA. Birds had free access to water throughout the trial. Body weight (BW) and uniformity (BW CV) were monitored. Boosters for infectious bronchitis and New Castle disease vaccines were administered at 18 WOA, and samples of pullets bled for antibody titer 5-day later. One pullet/pen was randomly selected, weighed, bled for plasma biochemistry, and necropsied for organ weights, ceca digesta for short-chain fatty acids (SCFA), and leg bones morphometry. In the starter and grower phases, birds fed HY were lighter and gained less (P < 0.05) than control birds. However, there were no diet effects (P > 0.05) on growth, the BW prelay phase, or BW uniformity throughout the trial. There were no (P > 0.05) diet effects on breast, gastrointestinal, liver and bursa weights, serum antibody titers, plasma biochemistry, SCFA and bone attributes. However, pullets fed HY had heavier (P = 0.047) spleen and tended to have lower (P = 0.080) plasma concentrations of aspartate aminotransferase (AST) relative to control pullets. In conclusion, the parameters assessed showed no negative consequences of feeding HY to broiler breeder pullets. However, effects on the spleen and plasma AST may indicate modest modulation of immunity and metabolism. The impact of the provision of HY during broiler breeder pullet phase on reproductive performance and chick quality should be investigated.

Graded levels of Eimeria infection linearly reduced the growth performance, altered the intestinal health, and delayed the onset of egg production of Hy-Line W-36 laying hens when infected at the prelay stage.

The aim of this experiment was to investigate how different levels of Eimeria infection affect the performance, intestinal health, oxidative status, and egg production of Hy-Line W-36 pullets and laying hens. Three hundred and sixty Hy-Line W-36 pullets, aged 15 wk, were randomly distributed into 5 treatment groups, each comprising 6 replicates and a nonchallenged control. At 15 wk, pullets were inoculated with different levels of mixed Eimeria species as high-dose, medium-high, medium-low, and low-dose treatments. The growth performance and average daily feed intake (ADFI) were measured from 0- to 18-days postinoculation (DPI), whereas hen day egg production (HDEP) was recorded from wk 19. The markers of gastrointestinal health and oxidative status were measured at 6 DPI, 14 DPI, and 23 wk of age. The findings revealed a significant linear reduction in growth performance in response to increased Eimeria challenge dosage on 6 and 14 DPI (P < 0.0001, P-L < 0.0001). An interaction between the graded level of Eimeria infection and DPI was observed for ADFI. The challenged pullets showed a reduction in ADFI starting at 4 DPI, which persisted until 14 DPI, when ADFI recovered back to normal. The most significant drop in feed intake was observed in 6 DPI in all the Eimeria-infected groups. The markers of gastrointestinal health (gastrointestinal permeability and tight junction proteins) were upregulated in challenged pullets because of infection, whereas the relative mRNA expression of key nutrient transporters was downregulated following infection on 6 and 14 DPI (P < 0.05). As a result of an infection on 6 DPI, the oxidative equilibrium was shifted toward the oxidative stress, and at the same time, upregulation of proinflammatory and inflammatory cytokines was observed (P < 0.05). An interaction between the Eimeria challenge dosage and bird age was observed for HDEP (P = 0.0427). The pullets infected with Eimeria started to lay eggs later than the Control birds. However, the HDEP of the challenged groups became similar to Control only at wk 22, 3 wk after laying eggs. In conclusion, coccidiosis reduced growth performance, altered gastrointestinal health, induced oxidative stress, and delayed egg production when infected at the prelay stage of pullets and negatively impacted the laying hens' overall performance.

Blocking Mitochondrial Pyruvate Transport Alters Corneal Myofibroblast Phenotype: A New Target for Treating Fibrosis.

Purpose: The purpose of this study was to critically test the hypothesis that mitochondrial pyruvate carrier (MPC) function is essential for maintenance of the corneal myofibroblast phenotype in vitro and in vivo. Methods: Protein and mRNA for canonical profibrotic markers were assessed in cultured cat corneal myofibroblasts generated via transforming growth factor (TGF)-ß1 stimulation and treated with either the thiazolidinedione (TZD) troglitazone or the MPC inhibitor alpha-cyano-beta-(1-phenylindol-3-yl) acrylate (UK-5099). RNA sequencing was used to gain insight into signaling modules related to instructive, permissive, or corollary changes in gene expression following treatment. A feline photorefractive keratectomy (PRK) model of corneal wounding was used to test the efficacy of topical troglitazone at reducing α-smooth muscle actin (SMA)-positive staining when applied 2 to 4 weeks postoperatively, during peak fibrosis. Results: Troglitazone caused cultured myofibroblasts to adopt a fibroblast-like phenotype through a noncanonical, peroxisome proliferator-activated receptor (PPAR)-γ-independent mechanism. Direct MPC inhibition using UK-5099 recapitulated this effect, but classic inhibitors of oxidative phosphorylation (OXPHOS) did not. Gene Set Enrichment Analysis (GSEA) of RNA sequencing data converged on energy substrate utilization and the Mitochondrial Permeability Transition pore as key players in myofibroblast maintenance. Finally, troglitazone applied onto an established zone of active fibrosis post-PRK significantly reduced stromal α-SMA expression. Conclusions: Our results provide empirical evidence that metabolic remodeling in myofibroblasts creates selective vulnerabilities beyond simply mitochondrial energy production, and that these are critical for maintenance of the myofibroblast phenotype. For the first time, we provide proof-of-concept data showing that this remodeling can be exploited to treat existing corneal fibrosis via inhibition of the MPC.

Fearfulness in commercial laying hens: a meta-analysis comparing brown and white egg layers.

High fearfulness in commercial laying hens can negatively affect production parameters and animal welfare. Brown and white egg layers differ in several behavioral characteristics, though reported differences in fearfulness are inconsistent. A meta-analysis was conducted to determine whether there are systematic differences in measures of fearfulness between brown and white layers. Twenty-three studies that examined either 1 or both of 2 behavioral tests were included: tonic immobility (TI) (longer duration = higher fearfulness, 16 studies) and novel object (NO) test (lower approach rate = higher fearfulness, 11 studies). The 2 tests were analyzed separately. TI analyses: A generalized linear mixed effect model (GLMM) with a lognormal distribution was fitted to describe the data with experiment nested in study as a random effect. Explanatory (X) variables were considered through backward selection, where potential X-variables included color (brown vs. white layers), decade (1980s, 2000s, 2020s), age (prelay vs. in lay), genetic stock (hybrid vs. grand-/parent stock), and methodology (back vs. side position). NO test analyses: univariable GLMMs with a beta distribution were fitted with approach rate as the Y-variable and color, decade, age, stock, or 2 methodological factors (test duration, single vs. group testing) as X-variables. Models were evaluated by assessing information criteria, residuals/random effect normality, significance of X-variables and model evaluation statistics (mean square prediction error, concordance correlation coefficient). TI duration was best explained by a color-by-decade interaction (P = 0.0006). Whites in the 1980s had longer TI durations (709.43 ± 143.88 s) than browns in the 1980s (282.90 ± 59.70 s), as well as in comparison to browns (208.80 ± 50.82 s) or whites (204.85 ± 49.60 s) in the 2020s. The NO approach rate was best explained by color (P ≤ 0.05 in 3 models), age (P < 0.05 in 3 models), and decade (P = 0.04). Whites had a higher approach rate (0.7 ± 0.07) than browns (0.5 ± 0.11), birds in lay a higher rate (0.8 ± 0.07) than birds prelay (0.4 ± 0.12), and approach rate for papers published in the 2000s (0.8 ± 0.09) was higher than in the 2020s (0.2 ± 0.12). The phylogenetic difference in the 1980s was no longer detectable after enforcing an upper limit on TI durations (10 min), as became common practice in later studies. Our findings suggest that phylogenetic differences in fearfulness and changes over time are test dependent, and this raises important questions and potential consequences for assessing hen welfare in commercial egg production.

Influence of the calcium and nutrient content of the prelay diet on egg production, egg quality, and tibiae mineralization of brown egg-laying hens from 16 to 63 wk of age.

The influence of the Ca and nutrient content of the diet fed from 16 to 19 wk of age, on egg production, egg quality, and tibiae mineralization, was studied in brown egg-laying hens from 16 to 63 wk of age. The experimental design was completely randomized with 4 prelay diets organized as a 2 × 2 factorial with 2 levels of Ca (2.5 vs. 3.8%) and 2 standardized ileal digestible Lys (g/kg) to AMEn (Mcal/kg) ratios (DLys:ME; 2.84 vs. 3.13) as main effects. From 20 to 63 wk of age, all hens received a common diet with 2.75 Mcal AMEn/kg, 0.75% DLys, and 3.8% Ca. Each treatment was replicated 18 times and the experimental unit was a cage with 10 hens. Hen production and egg quality traits were measured by period (4 wk), feeding phase (prelay and lay), and cumulatively (16-63 wk of age) and tibiae mineralization was measured at 63 wk of age. During the prelay phase, an increase in Ca delayed egg production (P = 0.065), reduced feed intake (P < 0.05), and increased BW gain (P < 0.01) and percentage of shell of the egg (P < 0.05). An increase in the DLys:ME ratio increased feed intake (P < 0.01) and reduced egg size (P < 0.01). Nutrient content of the prelay diets did not affect hen production during the lay phase, except egg weight that increased (P < 0.05) in hens previously fed the low DLys:ME ratio. Eggshell quality (weight, percentage, strength, and thickness) in this phase was better (P < 0.05) in hens previously fed 3.8% Ca. Cumulatively (16-63 wk of age), hens fed the high Ca prelay diets had better shell quality but tibiae mineralization was not affected. In conclusion, an increase in Ca content of the prelay diet from 2.5 to 3.8% improved shell quality for the entire egg cycle without showing any negative effect on hen production.

PPARγ activation inhibits endocytosis of claudin-4 and protects against deoxynivalenol-induced intestinal barrier dysfunction in IPEC-J2 cells and weaned piglets.

The role of peroxisome proliferator activated receptor gamma (PPARγ) in the regulation of adipocyte differentiation has been well characterized. Besides adipose tissue, PPARγ is also highly expressed in the intestine. However, the functional role of PPARγ in the regulation of intestinal function still remains poorly understood. In the present study, we sought to understand the role of PPARγ activation on regulation of intestinal barrier function in intestinal porcine epithelial cells (IPEC-J2) and weaned piglets exposed to the mycotoxin, deoxynivalenol (DON). PPARγ activation by rosiglitazone and troglitazone, two pharmacological PPARγ ligands, increased the protein expression of tight junction proteins (TJP), claudin-3 and 4. PPARγ inhibition increased endocytosis of claudin-4 which was reversed by its activation with troglitazone. DON exposure decreased the protein expression of TJP, and also significantly suppressed PPARγ transcriptional activity. Interestingly, PPARγ activation reversed the reduction of claudin-3 and 4 caused by DON in vitro and in vivo. PPARγ activation also partially restored the transepithelial electrical resistance (TEER) and reduced the permeability of fluorescein isothiocyanate-dextran (FITC-dextran) that have been negatively impacted by DON. These effects were lost in the presence of a specific PPARγ antagonist or in PPARγ knockout cells, confirming the importance of PPARγ in the regulation of intestinal barrier function and integrity. Likewise, in weaned pigs exposed to DON, the PPARγ agonist pioglitazone mitigated the impaired villus-crypt morphology caused by DON. Therefore, pharmacological and natural bioactive compounds with PPARγ stimulatory activities could be effective in preventing DON-induced gut barrier dysfunction.

Exploration of the Effects of TGF-ß Pathway-Based Pituitary Tumor of Rats on GH3 Cell Line after Intervention with Different Concentrations of TGZ.

The effect of the TGF-ß pathway-based pituitary tumor of rats on the GH3 cell line after intervention with different concentrations of troglitazone (TGZ) is explored. The CH3 cell line of 24 clean male SD rats with pituitary adenoma is selected. The cells are divided into a blank contrast set and an experimental set. The experimental set is divided into different TGZ concentration sets, including 1 × 10-3 TGZ set, 1 × 10-4 TGZ set, and 1 × 10-5 TGZ set. The cell proliferation is detected by the CCK-8 method, the protein expressions of CD147, TGF-ß1, and MMP-9 are detected by the western blot method, and the relative mRNA expressions of CD147, TGF-ß1, and MMP-9 are detected by the qRT-PCR method. The expression levels of CD147, TGF-ß1, and MMP-9 in CH3 cells of pituitary adenoma rats are notoriously lower, while the expression of CD147, TGF-31, and MMP-9 could be reduced by TGZ acting on the GH3 cell line. The specific mechanism of action of this effect on the invasive ability of GH3 cell lines is multifaceted, suggesting that peroxisome proliferator activator-receptor (PPAR-γ) agonists have good clinical application prospects in tumor therapy and can provide new targets and approaches for tumor drug therapy.

Influenza virus causes lung immunopathology through down-regulating PPARγ activity in macrophages.

Fatal influenza (flu) virus infection often activates excessive inflammatory signals, leading to multi-organ failure and death, also referred to as cytokine storm. PPARγ (Peroxisome proliferator-activated receptor gamma) agonists are well-known candidates for cytokine storm modulation. The present study identified that influenza infection reduced PPARγ expression and decreased PPARγ transcription activity in human alveolar macrophages (AMs) from different donors. Treatment with PPARγ agonist Troglitazone ameliorated virus-induced proinflammatory cytokine secretion but did not interfere with the IFN-induced antiviral pathway in human AMs. In contrast, PPARγ antagonist and knockdown of PPARγ in human AMs further enhanced virus-stimulated proinflammatory response. In a mouse model of influenza infection, flu virus dose-dependently reduced PPARγ transcriptional activity and decreased expression of PPARγ. Moreover, PPARγ agonist troglitazone significantly reduced high doses of influenza infection-induced lung pathology. In addition, flu infection reduced PPARγ expression in all mouse macrophages, including AMs, interstitial macrophages, and bone-marrow-derived macrophages but not in alveolar epithelial cells. Our results indicate that the influenza virus specifically targets the PPARγ pathway in macrophages to cause acute injury to the lung.

Lutein inhibits IL­6 expression by inducing PPAR­Î³ activation and SOCS3 expression in cerulein­stimulated pancreatic acinar cells.

Acute pancreatitis is a severe inflammatory disease of the pancreas. In experimental acute pancreatitis, cerulein induces the expression of interleukin­6 (IL­6) by activating Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 in pancreatic acinar cells. Ligands of peroxisome proliferator activated receptor­Î³ (PPAR­Î³) and suppressor of cytokine signaling (SOCS) 3 inhibit IL­6 expression by suppressing JAK2/STAT3 in cerulein­stimulated pancreatic acinar AR42J cells. Lutein, an oxygenated carotenoid, upregulates and activates PPAR­Î³ to regulate inflammation in a renal injury model. The present study aimed to determine whether lutein activated PPAR­Î³ and induced SOCS3 expression in unstimulated AR42J cells, and whether lutein inhibited activation of JAK2/STAT3 and IL­6 expression via activation of PPAR­Î³ and SOCS3 expression in cerulein­stimulated AR42J cells. The anti­inflammatory mechanism of lutein was determined using reverse transcription­quantitative PCR, western blot analysis and enzyme­linked immunosorbent assay in AR42J cells stimulated with or without cerulein. In another experiment, cells were treated with lutein and the PPAR­Î³ antagonist GW9662 or the PPAR­Î³ agonist troglitazone prior to cerulein stimulation to determine the involvement of PPAR­Î³ activation. The results indicated that lutein increased PPAR­Î³ and SOCS3 levels in unstimulated cells. Cerulein increased phospho­specific forms of JAK2 and STAT3, and mRNA and protein expression of IL­6, but decreased SOCS3 levels in AR42J cells. Cerulein­induced alterations were suppressed by lutein or troglitazone. GW9662 alleviated the inhibitory effect of lutein on JAK2/STAT3 activation and IL­6 expression in cerulein­stimulated cells. In conclusion, lutein inhibited the activation of JAK2/STAT3 and reduced IL­6 levels via PPAR­Î³­mediated SOCS3 expression in pancreatic acinar cells stimulated with cerulein.

Improvement of Oral Absorption of Poorly Water-Soluble Drugs by Solid Dispersions with Amphiphilic Phospholipid Polymer.

Solid dispersions are one of methods for solubilizing water-insoluble drugs. To enhance the bioavailability, maintenance of the supersaturated state and absorption of the dissolved drug in the gastrointestinal tract are important. We designed and synthesized amphiphilic 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymers as carriers for solid dispersions and evaluated the dissolution behavior in test solutions with different pH and additives. Solid dispersion of troglitazone with amphiphilic MPC copolymers having both aromatic rings and urethane bonds in the side chains showed rapid dissolution and excellent supersaturation maintenance. It was indicated that the balance between the interactions with drug molecules and the water affinity of the polymer should be considered when carriers for solid dispersions are designed. In addition, cell membrane permeability of the solid dispersion with the amphiphilic MPC copolymer was evaluated by the Dissolution / Permeation system, which consists of two liquid chambers and a monolayer of epithelial cells that mimics the intestinal dissolution and permeation process. Further, blood concentration of the drug when solid dispersions were orally administered in mice was also evaluated. The cell membrane permeability and oral absorbability were significantly improved, compared to the solid dispersions with poly(N-vinylpyrrolidone) and suspension or solution of crystalline troglitazone.

Sorghum (Sorghum bicolor) Extract-Induced Adipogenesis Is Independent of PPARγ Ser273 Phosphorylation in 3T3-L1 Adipocytes.

Previously we showed that the water-soluble fraction of sorghum extract (SE) improves adipogenesis in 3-isobutyl-1-methylxanthine (IBMX)/dexamethasone/insulin (MDI)/thiazolidinedione (TZD)-induced 3T3-L1 preadipocytes but downregulates genes related to peroxisome proliferator-activated receptor γ (PPARγ) and adipogenesis in both MDI- and MDI/TZD-induced 3T3-L1 adipocytes. In this study, we showed that SE treatment altered the accumulation of stained lipids in 3T3-L1 adipocytes induced by MDI/troglitazone (Tro). Immunoblot analyses indicated that SE treatment reduced adipocyte protein 2 (aP2) expression and induced peroxisome proliferator-activated receptor α (PPARα) protein expression in the presence of Tro in 3T3-L1 adipocytes. MDI/Tro treatment, but not MDI treatment, of 3T3-L1 cells induced PPARγ phosphorylation at Ser273. SE downregulated PPARγ expression in MDI-induced 3T3-L1 adipocytes and did not affect its phosphorylation at Ser273 in MDI- and MDI/Tro-induced 3T3-L1 adipocytes. Therefore, SE likely promotes adipogenesis and lipid metabolism in 3T3-L1 preadipocytes by cooperating with Tro independent of PPARγ Ser273 phosphorylation.

Troglitazone-Induced Autophagic Cytotoxicity in Lung Adenocarcinoma Cell Lines.

Lung cancer is the leading cause of cancer-related deaths worldwide. Troglitazone (TGZ), a peroxisome proliferator-activated receptor gamma (PPARγ) ligand, is a potential antitumor agent. However, the action mechanism of TGZ in lung adenocarcinoma cells has not been completely elucidated. To assess this mechanism and the anticancer effects of TGZ in human lung adenocarcinoma cell lines (A549 and H1975), we investigated the involvement of PPARγ, apoptosis, the mitogen-activated protein kinase (MAPK) pathway, protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway, and autophagy. Cell viability was measured using fluorescence-based assays. Apoptotic cells were detected by Hoechst 33342 and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining; protein expression was detected by Western blotting. TGZ inhibited cell proliferation in a dose-dependent manner in both cell lines, and the effect was not suppressed by a PPARγ inhibitor. Additionally, TGZ increased apoptotic cell number and upregulated p38 and c-Jun N-terminal kinase (JNK) phosphorylation; however, p38 and JNK inhibitors did not block TGZ-mediated inhibition of cell proliferation in either cell line. TGZ also upregulated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas an ERK1/2 inhibitor enhanced TGZ-mediated cytotoxicity in A549 cells. Additionally, TGZ increased LC3-II expression, and chloroquine (an autophagy inhibitor) attenuated TGZ-mediated inhibition of cell proliferation. These findings suggest that TGZ-induced inhibition of cell proliferation is PPARγ independent. TGZ-mediated inhibition of cell proliferation was accompanied by apoptosis and independent of the MAPK signaling pathway. These results suggest that TGZ inhibits cell proliferation through autophagy-induced cytotoxicity. This study demonstrated that chemotherapy using TGZ may be effective for lung adenocarcinoma.

Advancing the Adverse Outcome Pathway for PPARγ Inactivation Leading to Pulmonary Fibrosis Using Bradford-Hill Consideration and the Comparative Toxicogenomics Database.

Pulmonary fibrosis is regulated by transforming growth factor-ß (TGF-ß) and peroxisome proliferator-activated receptor-gamma (PPARγ). An adverse outcome pathway (AOP) for PPARγ inactivation leading to pulmonary fibrosis has been previously developed. To advance the development of this AOP, the confidence of the overall AOP was assessed using the Bradford-Hill considerations as per the recommendations from the Organisation for Economic Co-operation and Development (OECD) Users' Handbook. Overall, the essentiality of key events (KEs) and the biological plausibility of key event relationships (KERs) were rated high. In contrast, the empirical support of KERs was found to be moderate. To experimentally evaluate the KERs from the molecular initiating event (MIE) and KE1, PPARγ (MIE) and TGF-ß (KE1) inhibitors were used to examine the effects of downstream events following inhibition of their upstream events. PPARγ inhibition (MIE) led to TGF-ß activation (KE1), upregulation in vimentin expression (KE3), and an increase in the fibronectin level (KE4). Similarly, activated TGF-ß (KE1) led to an increase in vimentin (KE3) and fibronectin expression (KE4). In the database analysis using the Comparative Toxicogenomics Database, 31 genes related to each KE were found to be highly correlated with pulmonary fibrosis, and the top 21 potential stressors were suggested. The AOP for pulmonary fibrosis evaluated in this study will be the basis for the screening of inhaled toxic substances in the environment.

Troglitazone inhibits hepatic oval cell proliferation by inducing cell cycle arrest through Hippo/YAP pathway regulation.

Hepatic oval cells have strong proliferation and differentiation capabilities and are activated when chronic liver injury occurs or when liver function is severely impaired. Peroxisome proliferation-activated receptors (PPARs) are ligand-dependent, sequence-specific nuclear transcription factors. PPARγ is closely related to liver diseases (such as liver cancer, liver fibrosis and non-alcoholic fatty liver disease). As the main effector downstream of the Hippo signaling pathway, YAP can activate the hepatic progenitor cell program, and different expression or activity levels of YAP can determine different liver cell fates. We found that troglitazone (TRO), a classic PPARγ activator, can inhibit the growth of hepatic oval cells, and flow cytometry results showed that TRO inhibited the growth of WB-F344 cells by arresting the cells in the G0/1 phase. Western blot results also confirmed changes in G0/1 phase-related protein expression. Further experiments showed that PPARγ agonists induced hepatic oval cell proliferation inhibition and cell cycle G0/1 phase arrest through the Hippo/YAP pathway. Our experiment demonstrated, for the first time, the relationship between PPARγ and the Hippo/YAP pathway in liver oval cells and revealed that PPARγ acts as a negative regulator of liver regeneration by inhibiting the proliferation of oval cells.

Permeabilized Cryopreserved Human Hepatocytes as an Exogenous Metabolic System in a Novel Metabolism-Dependent Cytotoxicity Assay for the Evaluation of Metabolic Activation and Detoxification of Drugs Associated with Drug-Induced Liver Injuries: Results with Acetaminophen, Amiodarone, Cyclophosphamide, Ketoconazole, Nefazodone, and Troglitazone.

We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system, and human embryonic kidney 293 (HEK293) cells, a cell line devoid of drug-metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, uridine 5'-diphosphoglucuronic acid (UDPGA) for uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated glucuronidation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for cytosolic sulfotransferase (SULT) mediated sulfation, and glutathione (GSH) for glutathione S-transferase (GST) mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone, and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH/NAD+, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole, and troglitazone, by PAPS for acetaminophen, ketoconazole, and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied toward the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. SIGNIFICANCE STATEMENT: Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential and to aid the assessment of metabolism-based risk factors. Glutathione (GSH) detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites that may be applied toward the evaluation of idiosyncratic hepatotoxicity.

NTCP Oligomerization Occurs Downstream of the NTCP-EGFR Interaction during Hepatitis B Virus Internalization.

Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and noncompetitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, coimmunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affects the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms. IMPORTANCE Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.

Role of respiratory uncoupling in drug-induced mitochondrial permeability transition.

Mitochondrial injury contributes to severe drug-induced liver injury. Particularly, mitochondrial permeability transition (MPT) is thought to be relevant to cytolytic hepatitis. However, the mechanism of drug-induced MPT is unclear and prediction of MPT is not adequately evaluated in the preclinical stage. In a previous study, we found that troglitazone, a drug withdrawn due to liver injury, induced MPT via mild depolarization probably resulting from uncoupling. Herein, we investigated whether other drugs that induce MPT share similar properties as troglitazone, using isolated mitochondria from rat liver. Of the 22 test drugs examined, six drugs, including troglitazone, induced MPT and showed an uncoupling effect. Additionally, receiver operating characteristic analysis was conducted to predict the MPT potential from the respiratory control ratio, an indicator of uncoupling intensity. Results showed that 2.5 was the best threshold that exhibited high sensitivity (1.00) and high specificity (0.81), indicating that uncoupling was correlated with MPT potential. Activation of calcium-independent phospholipase A2 appeared to be involved in uncoupling-induced MPT. Furthermore, a strong relationship between MPT intensity and the uncoupling effect among similar compounds was confirmed. These results may help in predicting MPT potential using cultured cells and modifying the chemical structures of the drugs to reduce MPT risk.

CD73 induces gemcitabine resistance in pancreatic ductal adenocarcinoma: A promising target with non-canonical mechanisms.

CD73, a cell surface-localized ecto-5'-nucleotidase, is the major enzymatic source of extracellular adenosine. Canonically, it plays multiple roles in cancer-related processes via its metabolite. As a druggable target, clinical trials targeting CD73 in various malignant diseases are currently ongoing. Here, we report the ecto-5'-nucleotidase-independent functions of CD73 in pancreatic ductal adenocarcinoma (PDAC). Our findings support that the elevated expression of CD73 in PDAC cells promotes gemcitabine (GEM) resistance by activating AKT. We discovered that a large amount of intracellular CD73 are localized in the endoplasmic reticulum membrane. Intracellular CD73 physically interacts with major vault protein to activate the SRC-AKT circuit. Troglitazone (TGZ) is a peroxisome proliferator-activated receptor gamma agonist that could inhibit the expression of CD73. The administration of TGZ markedly enhances sensitivity to GEM via downregulating CD73 in PDAC. Our findings support that CD73 could be targeted to overcome chemoresistance in PDAC.

The antidiabetic drug troglitazone protects against PrP (106­126)­induced neurotoxicity via the PPARγ­autophagy pathway in neuronal cells.

Prion diseases, which involve the alteration of cellular prion protein into a misfolded isoform, disrupt the central nervous systems of humans and animals alike. Prior research has suggested that peroxisome proliferator­activator receptor (PPAR)γ and autophagy provide some protection against neurodegeneration. PPARs are critical to lipid metabolism regulation and autophagy is one of the main cellular mechanisms by which cell function and homeostasis is maintained. The present study examined the effect of troglitazone, a PPARγ agonist, on autophagy flux in a prion peptide (PrP) (106­126)­mediated neurodegeneration model. Western blot analysis confirmed that treatment with troglitazone increased LC3­II and p62 protein expression, whereas an excessive increase in autophagosomes was verified by transmission electron microscopy. Troglitazone weakened PrP (106­126)­mediated neurotoxicity via PPARγ activation and autophagy flux inhibition. A PPARγ antagonist blocked PPARγ activation as well as the neuroprotective effects induced by troglitazone treatment, indicating that PPARγ deactivation impaired troglitazone­mediated protective effects. In conclusion, the present study demonstrated that troglitazone protected primary neuronal cells against PrP (106­126)­induced neuronal cell death by inhibiting autophagic flux and activating PPARγ signals. These results suggested that troglitazone may be a useful therapeutic agent for the treatment of neurodegenerative disorders and prion diseases.

Performance of preclinical models in predicting drug-induced liver injury in humans: a systematic review.

Drug-induced liver injury (DILI) causes one in three market withdrawals due to adverse drug reactions, causing preventable human suffering and massive financial loss. We applied evidence-based methods to investigate the role of preclinical studies in predicting human DILI using two anti-diabetic drugs from the same class, but with different toxicological profiles: troglitazone (withdrawn from US market due to DILI) and rosiglitazone (remains on US market). Evidence Stream 1: A systematic literature review of in vivo studies on rosiglitazone or troglitazone was conducted (PROSPERO registration CRD42018112353). Evidence Stream 2: in vitro data on troglitazone and rosiglitazone were retrieved from the US EPA ToxCast database. Evidence Stream 3: troglitazone- and rosiglitazone-related DILI cases were retrieved from WHO Vigibase. All three evidence stream analyses were conducted according to evidence-based methodologies and performed according to pre-registered protocols. Evidence Stream 1: 9288 references were identified, with 42 studies included in analysis. No reported biomarker for either drug indicated a strong hazard signal in either preclinical animal or human studies. All included studies had substantial limitations, resulting in "low" or "very low" certainty in findings. Evidence Stream 2: Troglitazone was active in twice as many in vitro assays (129) as rosiglitazone (60), indicating a strong signal for more off-target effects. Evidence Stream 3: We observed a fivefold difference in both all adverse events and liver-related adverse events reported, and an eightfold difference in fatalities for troglitazone, compared to rosiglitazone. In summary, published animal and human trials failed to predict troglitazone's potential to cause severe liver injury in a wider patient population, while in vitro data showed marked differences in the two drugs' off-target activities, offering a new paradigm for reducing drug attrition in late development and in the market. This investigation concludes that death and disability due to adverse drug reactions may be prevented if mechanistic information is deployed at early stages of drug development by pharmaceutical companies and is considered by regulators as a part of regulatory submissions.