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At least 199 records · Page 11
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Prevention of acute liver injury by suppressing plasma kallikrein-dependent activation of latent TGF-β

Highlights: • TGF-β contributes to the pathogenesis of acute liver injury (ALI). • PLK-dependent TGF-β activation occurs in/around macrophages in ALI mice. • R58 latency associated protein-degradation products serve as an indicator for ALI. • Inhibiting PLK suppresses apoptosis and improves survival rate of ALI mice. Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor β (TGF-β) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-β is activated partly through proteolytic cleavage by a serine protease plasma kallikrein (PLK) between the R58 and L59 residues of its propeptide region. Recently, we developed a specific monoclonal antibody to detect the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs) that reflect PLK-dependent TGF-β activation. This study aimed to explore the potential roles of PLK-dependent TGF-β activation in the pathogenesis of ALI. We established a mouse ALI model via the injection of anti-Fas antibodies (Jo2) and observed increases in the TGF-β1 mRNA level, Smad3 phosphorylation, TUNEL-positive apoptotic hepatocytes and R58-positive cells in the liver tissues of Jo2-treated mice. The R58 LAP-DPs were observed in/around F4/80-positive macrophages, while macrophage depletion with clodronate liposomes partly alleviated the Jo2-induced liver injury. Blocking PLK-dependent TGF-β activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-β receptor-mediated signaling pathway using SB431542 significantly prevented Jo2-induced hepatic apoptosis and mortality. Furthermore, similar phenomena were observed in the mouse model of ALI with the administration of acetaminophen (APAP). In summary, R58 LAP-DPs reflecting PLK-dependent TGF-β activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-β activation has potential as a therapeutic strategy for ALI.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Hepatic conditional knockout of ATF6 exacerbates liver metabolic damage by repressing autophage through MTOR pathway

Highlights: • Conditional knockout of ATF6 exacerbates liver metabolic damage. • Conditional knockout of ATF6 represses autophage. • Conditional knockout of ATF6 activates MTOR pathway. The liver is a central metabolic organ. Activating transcription factor 6 (ATF6) acts as an ER stress responsive gene and is reported to attenuate hepatic steatosis. Over expressing a dominant-negative form of ATF6 exacerbates glucose intolerance and insulin resistance. In the present study, we used the conditional knockout technique to specifically knockout ATF6 in the mouse liver. We used qPCR to detect the mRNA levels of related genes. Western blot analysis was used to evaluate protein levels. Flow cytometry assay showed the apoptosis status. Glucose tolerance tests and insulin tolerance tests were used to determine glucose and insulin sensitivity. The results showed that liver specific knockout of ATF6 exacerbated HFD-induced hepatic steatosis and glucose tolerance. Abolished ATF6 exacerbated gluconeogenic metabolism by MTOR mediated down regulation of autophage. In conclusion, these findings suggest that therapeutic strategies by supplementing ATF6 may be beneficial for the treatment of glucose intolerance as well as insulin resistance in the high fat induced liver metabolic damage condition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Liraglutide protects non-alcoholic fatty liver disease via inhibiting NLRP3 inflammasome activation in a mouse model induced by high-fat diet

Liraglutide, a glucagon-like peptide-1 (GLP-1) analogue that has recently become the first-line treatment for type 2 diabetes mellitus (T2DM), has also been reported to decrease fatty degeneration of the liver. The purpose of this study is to explore whether liraglutide improves high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) in mice through inhibiting the NLRP3 inflammasome in the liver. After daily intraperitoneal injection of liraglutide (0.6 mg/kg body weight) for four weeks, the liver, liver/body weight, serum levels of ALT, AST, total cholesterol, triglycerides and LDL were significantly decreased in a high-fat diet-induced NAFLD mouse model. The hepatic steatosis among sections of H&E and Oil Red O staining was also markedly reduced after treatment with liraglutide. The expressions of NLRP3 inflammasome components (including NLRP3, ASC, and caspase-1) in the liver of mice after treatment with liraglutide were decreased substantially. In vitro studies found that the mitochondrial dysfunction in Kupffer cells induced by palmitic acid was attenuated, and the protein levels of NLRP3, ASC and caspase-1 were also decrease markedly. These results demonstrate that liraglutide was able to alleviate high-fat diet-induced hepatic steatosis via inhibiting NLRP3 inflammasome activation, suggesting that liraglutide is a potent drug that can reverse the pathological hallmarks of NAFLD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Magnetic Resonance Imaging–Guided versus Surrogate-Based Motion Tracking in Liver Radiation Therapy: A Prospective Comparative Study

Purpose: This study applied automatic feature detection on cine–magnetic resonance imaging (MRI) liver images in order to provide a prospective comparison between MRI-guided and surrogate-based tracking methods for motion-compensated liver radiation therapy. Methods and Materials: In a population of 30 subjects (5 volunteers plus 25 patients), 2 oblique sagittal slices were acquired across the liver at high temporal resolution. An algorithm based on scale invariant feature transform (SIFT) was used to extract and track multiple features throughout the image sequence. The position of abdominal markers was also measured directly from the image series, and the internal motion of each feature was quantified through multiparametric analysis. Surrogate-based tumor tracking with a state-of-the-art external/internal correlation model was simulated. The geometrical tracking error was measured, and its correlation with external motion parameters was also investigated. Finally, the potential gain in tracking accuracy relying on MRI guidance was quantified as a function of the maximum allowed tracking error. Results: An average of 45 features was extracted for each subject across the whole liver. The multi-parametric motion analysis reported relevant inter- and intrasubject variability, highlighting the value of patient-specific and spatially-distributed measurements. Surrogate-based tracking errors (relative to the motion amplitude) were were in the range 7% to 23% (1.02-3.57mm) and were significantly influenced by external motion parameters. The gain of MRI guidance compared to surrogate-based motion tracking was larger than 30% in 50% of the subjects when considering a 1.5-mm tracking error tolerance. Conclusions: Automatic feature detection applied to cine-MRI allows detailed liver motion description to be obtained. Such information was used to quantify the performance of surrogate-based tracking methods and to provide a prospective comparison with respect to MRI-guided radiation therapy, which could support the definition of patient-specific optimal treatment strategies.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2016

Time-Resolved Intrafraction Target Translations and Rotations During Stereotactic Liver Radiation Therapy: Implications for Marker-based Localization Accuracy

Purpose: Image guided liver stereotactic body radiation therapy (SBRT) often relies on implanted fiducial markers. The target localization accuracy decreases with increased marker-target distance. This may occur partly because of liver rotations. The aim of this study was to examine time-resolved translations and rotations of liver marker constellations and investigate if time-resolved intrafraction rotational corrections can improve localization accuracy in liver SBRT. Methods and Materials: Twenty-nine patients with 3 implanted markers received SBRT in 3 to 6 fractions. The time-resolved trajectory of each marker was estimated from the projections of 1 to 3 daily cone beam computed tomography scans and used to calculate the translation and rotation of the marker constellation. In all cone beam computed tomography projections, the time-resolved position of each marker was predicted from the position of another surrogate marker by assuming that the marker underwent either (1) the same translation as the surrogate marker; or (2) the same translation as the surrogate marker corrected by the rotation of the marker constellation. The localization accuracy was quantified as the root-mean-square error (RMSE) between the estimated and the actual marker position. For comparison, the RMSE was also calculated when the marker's position was estimated as its mean position for all the projections. Results: The mean translational and rotational range (2nd-98th percentile) was 2.0 mm/3.9° (right-left), 9.2 mm/2.9° (superior-inferior), 4.0 mm/4.0° (anterior-posterior), and 10.5 mm (3-dimensional). Rotational corrections decreased the mean 3-dimensional RMSE from 0.86 mm to 0.54 mm (P<.001) and halved the RMSE increase per millimeter increase in marker distance. Conclusions: Intrafraction rotations during liver SBRT reduce the accuracy of marker-guided target localization. Rotational correction can improve the localization accuracy with a factor of approximately 2 for large marker-target distances.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2015

Application of modified dynamic conformal arc (MDCA) technique on liver stereotactic body radiation therapy (SBRT) planning following RTOG 0438 guideline

Liver stereotactic body radiation therapy (SBRT) is a feasible treatment method for the nonoperable, patient with early-stage liver cancer. Treatment planning for the SBRT is very important and has to consider the simulation accuracy, planning time, treatment efficiency effects etc. The modified dynamic conformal arc (MDCA) technique is a 3-dimensional conformal arc planning method, which has been proposed for liver SBRT planning at our center. In this study, we compared the MDCA technique with the RapidArc technique in terms of planning target volume (PTV) coverage and sparing of organs at risk (OARs). The results show that the MDCA technique has comparable plan quality to RapidArc considering PTV coverage, hot spots, heterogeneity index, and effective liver volume. For the 5 PTVs studied among 4 patients, the MDCA plan, when compared with the RapidArc plan, showed 9% more hot spots, more heterogeneity effect, more sparing of OARs, and lower liver effective volume. The monitor unit (MU) number for the MDCA plan is much lower than for the RapidArc plans. The MDCA plan has the advantages of less planning time, no-collision treatment, and a lower MU number.

61 RADIATION PROTECTION AND DOSIMETRY↗
DOE OSTI2013

Diphenylarsinic acid, a chemical warfare-related neurotoxicant, promotes liver carcinogenesis via activation of aryl hydrocarbon receptor signaling and consequent induction of oxidative DAN damage in rats

Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. Inorganic arsenic is carcinogenic in humans and its organic arsenic metabolites are carcinogenic in animal studies, raising serious concerns about the carcinogenicity of DPAA. However, the carcinogenic potential of DPAA has not yet been evaluated. In the present study we found that DPAA significantly enhanced the development of diethylnitrosamine-induced preneoplastic lesions in the liver in a medium-term rat liver carcinogenesis assay. Evaluation of the expression of cytochrome P450 (CYP) enzymes in the liver revealed that DPAA induced the expression of CYP1B1, but not any other CYP1, CYP2, or CYP3 enzymes, suggesting that CYP1B1 might be the enzyme responsible for the metabolic activation of DPAA. We also found increased oxidative DNA damage, possibly due to elevated CYP1B1 expression. Induction of CYP1B1 has generally been linked with the activation of AhR, and we found that DPAA activates the aryl hydrocarbon receptor (AhR). Importantly, the promotion effect of DPAA was observed only at a dose that activated the AhR, suggesting that activation of AhR and consequent induction of AhR target genes and oxidative DNA damage plays a vital role in the promotion effects of DPAA. The present study provides, for the first time, evidence regarding the carcinogenicity of DPAA and indicates the necessity of comprehensive evaluation of its carcinogenic potential using long-term carcinogenicity studies. - Highlights: • DPAA, an environmental neurotoxicant, promotes liver carcinogenesis in rats. • DPAA is an activator of AhR signaling pathway. • DPAA promoted oxidative DNA damage in rat livers. • AhR target gene CYP 1B1 might be involved in the metabolism of DPAA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Plasma biomarkers of liver injury and inflammation demonstrate a lack of apoptosis during obstructive cholestasis in mice

Cholestasis is a pathological common component of numerous liver diseases that results in hepatotoxicity, inflammation, and cirrhosis when untreated. While the predominant hypothesis in cholestatic liver injury remains hepatocyte apoptosis due to direct toxicity of hydrophobic bile acid exposure, recent work suggests that the injury occurs through inflammatory necrosis. In order to resolve this controversy, we used novel plasma biomarkers to assess the mechanisms of cell death during early cholestatic liver injury. C57Bl/6 mice underwent bile duct ligation (BDL) for 6–72 h, or sham operation. Another group of mice were given D-galactosamine and endotoxin as a positive control for apoptosis and inflammatory necrosis. Plasma levels of full length cytokeratin-18 (FL-K18), microRNA-122 (miR-122) and high mobility group box-1 protein (HMGB1) increased progressively after BDL with peak levels observed after 48 h. These results indicate extensive cell necrosis after BDL, which is supported by the time course of plasma alanine aminotransferase activities and histology. In contrast, plasma caspase-3 activity, cleaved caspase-3 protein and caspase-cleaved cytokeratin-18 fragments (cK18) were not elevated at any time during BDL suggesting the absence of apoptosis. In contrast, all plasma biomarkers of necrosis and apoptosis were elevated 6 h after Gal/End treatment. In addition, acetylated HMGB1, a marker for macrophage and monocyte activation, was increased as early as 12 h but mainly at 48–72 h. However, progressive neutrophil accumulation in the area of necrosis started at 6 h after BDL. In conclusion, these data indicate that early cholestatic liver injury in mice is an inflammatory event, and occurs through necrosis with little evidence for apoptosis. - Highlights: • The mechanism of cell death during cholestasis remains a controversial topic. • Plasma biomarkers offer new insight into cell death after bile duct ligation. • Cytokeratin-18, microRNA-122 and HMGB1 levels implicate necrosis. • Acetylated HMGB1 levels rise late after BDL confirming inflammation. • BDL-induced liver injury involves mainly inflammation and necrosis but no apoptosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B{sub 1} and its dependence on p53 genotype

Aflatoxin B{sub 1} (AFB{sub 1}) is biotransformed in vivo into an epoxide metabolite that forms DNA adducts that may induce cancer if not repaired. p53 is a tumor suppressor gene implicated in the regulation of global nucleotide excision repair (NER). Male heterozygous p53 knockout (B6.129-Trp53{sup tm1Brd}N5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB{sub 1} for 26 weeks. NER activity was assessed with an in vitro assay, using AFB{sub 1}-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB{sub 1}–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposed to 0.2 ppm and 1.0 ppm AFB{sub 1} respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05). In heterozygous p53 knockout mice, repair of AFB{sub 1}–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB{sub 1} or in liver extracts from mice treated with either AFB{sub 1} concentration. p53 genotype did not affect basal levels of repair. AFB{sub 1} exposure did not alter repair of AFB{sub 1}-derived formamidopyrimidine adducts in lung or liver extracts of either mouse genotype nor did it affect XPA or XPB protein levels. In summary, chronic exposure to AFB{sub 1} increased NER activity in wild-type mice, and this response was diminished in heterozygous p53 knockout mice, indicating that loss of one allele of p53 limits the ability of NER to be up-regulated in response to DNA damage. - Highlights: • Mice are chronically exposed to low doses of the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}). • The effects of AFB{sub 1} and p53 status on nucleotide excision repair are investigated. • AFB{sub 1} increases nucleotide excision repair in wild type mouse lung and liver. • This increase is attenuated in p53 heterozygous mouse lung and liver. • Results portray the role of p53 in nucleotide excision repair after AFB{sub 1} exposure.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Experimental models of hepatotoxicity related to acute liver failure

Acute liver failure can be the consequence of various etiologies, with most cases arising from drug-induced hepatotoxicity in Western countries. Despite advances in this field, the management of acute liver failure continues to be one of the most challenging problems in clinical medicine. The availability of adequate experimental models is of crucial importance to provide a better understanding of this condition and to allow identification of novel drug targets, testing the efficacy of new therapeutic interventions and acting as models for assessing mechanisms of toxicity. Experimental models of hepatotoxicity related to acute liver failure rely on surgical procedures, chemical exposure or viral infection. Each of these models has a number of strengths and weaknesses. This paper specifically reviews commonly used chemical in vivo and in vitro models of hepatotoxicity associated with acute liver failure. - Highlights: • The murine APAP model is very close to what is observed in patients. • The Gal/ET model is useful to study TNFα-mediated apoptotic signaling mechanisms. • Fas receptor activation is an effective model of apoptosis and secondary necrosis. • The ConA model is a relevant model of auto-immune hepatitis and viral hepatitis. • Multiple time point evaluation needed in experimental models of acute liver injury.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Tea polyphenols EGCG and TF restrict tongue and liver carcinogenesis simultaneously induced by N-nitrosodiethylamine in mice

The aim of this study is to understand the molecular mechanisms of N-nitrosodiethylamine (NDEA) induced multi-organ carcinogenesis in tongue and liver of the same mouse and restriction of carcinogenesis by Epigallocatechin gallate (EGCG) and Theaflavin (TF), if any. For that purpose, cellular proliferation/apoptosis, prevalence of CD44 positive stem cell population and expressions of some key regulatory genes of self renewal Wnt and Hedgehog (Hh) pathways and some of their associated genes were analyzed in the NDEA induced tongue and liver lesions in absence or presence of EGCG/TF. Chronic NDEA exposure in oral cavity could decrease mice body weights and induce tongue and liver carcinogenesis with similar histological stages (severe dysplasia up to 30th weeks of NDEA administration). Increasing mice body weights were seen in continuous and post EGCG/TF treated groups. EGCG/TF treatment could restrict both the carcinogenesis at similar histological stages showing potential chemopreventive effect in continuous treated groups (mild dysplasia) followed by pre treatment (moderate dysplasia) and therapeutic efficacy in post treated groups (mild dysplasia) up to 30th week. The mechanism of carcinogenesis by NDEA and restriction by the EGCG/TF in both tongue and liver were similar and found to be associated with modulation in cellular proliferation/apoptosis and prevalence of CD44 positive population. The up-regulation of self renewal Wnt/β-catenin, Hh/Gli1 pathways and their associated genes Cyclin D1, cMyc and EGFR along with down regulation of E-cadherin seen during the carcinogenesis processes were found to be modulated during the restriction processes by EGCG/TF. - Highlights: • Simultaneous tongue and liver carcinogenesis in mice by oral NDEA administration • Restriction of both carcinogenesis by EGCG and TF at early pre-malignant stages • The mechanisms of carcinogenesis and restriction were similar in both the organs. • Changes in proliferation/apoptosis and CD44 + ve population were seen in the events. • The self renewal Wnt and Hedgehog pathways were modulated during the restriction.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Sub-chronic 90-day toxicity of neamine in SD rats and its anti-liver cancer activity in vitro and in vivo

Neamine, an inhibitor of angiogenin (ANG), is a new investigative anticancer drug currently in preclinical stage. Here we report the 90-day sub-chronic toxicity of neamine in SD rats and its anti-liver cancer activity in vitro and in vivo. Neamine has a No Observed Adverse Effect Level (NOAEL) of 12 and 16 mg·kg{sup −1}·d{sup −1} for female and male rats, respectively. No mortality was found. The adverse effects included increased organ coefficients of spleen and kidney, increased BUN in both female and male rats at high dose, increased CR and decreased organ coefficients of heart and liver in male rats at high dose. All of which, except the kidney coefficient and BUN in males, returned to normal levels after 28-day recovery. Histopathological examination revealed vacuolar degeneration of glomerulus, degeneration of renal tubules and cast in the kidneys, which were also recovered except in males of high-dosing group. These results indicate that kidney is the most susceptible organ for neamine toxicity. Tissue microarray analysis validated that ANG is up-regulated in hepatocellular carcinoma accompanied by increased nuclear translocation, suggesting that ANG is a possible target for drug development in liver cancer treatment. Neamine blocked nuclear translocation of ANG in HUVEC and HepG2 cells, and inhibited ANG-stimulated cell proliferation without affecting basal level cell proliferation. Neamine also inhibited progression of HepG2 xenografts in athymic mice accompanied by decreased angiogenesis and cancer cell proliferation. These results suggest that neamine is a specific ANG inhibitor with low toxicity and high anti-liver cancer efficacy. - Highlights: • The NOAEL of neamine is 12 mg·kg{sup −1}·d{sup −1} for females and 16 mg·kg{sup −}1·d{sup −1} for males. • The most susceptible organ for neamine toxicity is kidney. • Neamine inhibits the progression of xenograft HepG2 liver cancer in athymic mice.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Perfluorooctanoic acid exposure disturbs glucose metabolism in mouse liver

Environmental pollutants such as perfluorooctanoic acid (PFOA) can influence human metabolism processes and are associated with certain metabolic diseases. To investigate the effect of PFOA on liver glucose homeostasis, adult male Balb/c mice were orally administered 1.25 mg/kg of PFOA for 28 d consecutively. Compared with the control mice, the body weights of the PFOA-treated mice were unchanged following exposure. However, PFOA exposure increased fasting blood glucose levels and decreased glycogen and glucose content in the liver of treated mice, but did not influence blood insulin significantly. The increased blood glucagon might contribute to the hyperglycemia observed in the PFOA-treated group compared with the control group. In addition, pyruvate tolerance tests supported enhanced glucose production ability in PFOA-exposed mice. Consistent with the increase in blood glucose and decrease in hepatic glucose and glycogen, PFOA exposure decreased the protein level of glycogen synthase in the mouse liver, but increased the level of glucokinase. Furthermore, liver pyruvate, as well as mRNA levels of enzymes involved in the Krebs cycle, such as citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase, increased in the PFOA-treated group. PFOA exposure did not affect muscle glucose or glycogen levels. Indirect calorimetry showed higher VO{sub 2} consumption and respiratory quotient values in the PFOA-treated group compared with the control group, implying that PFOA treatment might promote energy consumption in mice, with a reliance on carbohydrates as a primary source of energy. Thus, our findings indicate that subacute exposure to PFOA might enhance glycogenolysis and gluconeogenesis and promote carbohydrate consumption. - Highlights: • PFOA exposure induced high fasting blood glucose. • PFOA exposure decreased glycogen and glucose content in the liver. • PFOA exposure enhanced glycogenolysis and gluconeogenesis. • PFOA exposure promoted carbohydrate consumption.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival

Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24 h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. - Highlights: • Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes. • Hepatocyte nuclear factor 4A (HNF4A) plays a role in hepatic differentiation. • HNF4A improved hepatic differentiation of IHH. • Transplantation of HNF4A-overexpressing IHH could improve liver function.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Mechanism of TRIM25 mediated ubiquitination of metastasis associated protein (MTA) 1 in normal liver cells

Ninety percent of all cancer related deaths happen due to metastatic progression. One important protein facilitating metastatic progression in hepatocellular carcinoma (HCC) is the metastasis associated 1 protein (MTA-1). We have earlier shown that in the context of HCC and normal liver cell lines, HuH6 and THLE-2, respectively. MTA-1 protein is actively stabilized in HCC cell lines and actively degraded in normal liver cells. We had also shown that TRIM25 is the E3 ligase that interacts with and degrades MTA-1 protein in normal liver cells. However, the exact mechanism by which TRIM25 degrades MTA-1 protein has still not been elucidated. In the study, we used both in situ prediction algorithms and mass spectrometry based post-translational modification analysis to map the lysine residues in MTA-1 that are polyubiquitinated. Whereas UbPred algorithm revealed a combination of medium and low confidence sites, it revealed only one high confidence lysine (K98) residue. The hCKSAAP{sub U}bSite algorithm also predicted K98 site. Mass spectrometry analysis also showed that K98 has ubiquitin modification. Immunofluorescence analysis showed that in normal liver cell line, THLE-2, which has high expression of TRIM25, ectopically expressed FLAG-tagged wild-type MTA-1 was actively degraded, but the K98R mutant MTA-1 was not. In vitro ubiquitination assay using recombinant wild-type and K98R mutant MTA-1 confirmed that MTA-1 is poly-ubiquitinated at K98 residue by TRIM25. The K98R mutant had a longer half-life than wild-type MTA-1 protein in an in vitro protein stability assay. We establish that TRIM25 ubiquitinates MTA-1 at lysine 98 and degrades it normal liver cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Three-dimensional liver motion tracking using real-time two-dimensional MRI

Purpose: Combined magnetic resonance imaging (MRI) systems and linear accelerators for radiotherapy (MR-Linacs) are currently under development. MRI is noninvasive and nonionizing and can produce images with high soft tissue contrast. However, new tracking methods are required to obtain fast real-time spatial target localization. This study develops and evaluates a method for tracking three-dimensional (3D) respiratory liver motion in two-dimensional (2D) real-time MRI image series with high temporal and spatial resolution. Methods: The proposed method for 3D tracking in 2D real-time MRI series has three steps: (1) Recording of a 3D MRI scan and selection of a blood vessel (or tumor) structure to be tracked in subsequent 2D MRI series. (2) Generation of a library of 2D image templates oriented parallel to the 2D MRI image series by reslicing and resampling the 3D MRI scan. (3) 3D tracking of the selected structure in each real-time 2D image by finding the template and template position that yield the highest normalized cross correlation coefficient with the image. Since the tracked structure has a known 3D position relative to each template, the selection and 2D localization of a specific template translates into quantification of both the through-plane and in-plane position of the structure. As a proof of principle, 3D tracking of liver blood vessel structures was performed in five healthy volunteers in two 5.4 Hz axial, sagittal, and coronal real-time 2D MRI series of 30 s duration. In each 2D MRI series, the 3D localization was carried out twice, using nonoverlapping template libraries, which resulted in a total of 12 estimated 3D trajectories per volunteer. Validation tests carried out to support the tracking algorithm included quantification of the breathing induced 3D liver motion and liver motion directionality for the volunteers, and comparison of 2D MRI estimated positions of a structure in a watermelon with the actual positions. Results: Axial, sagittal, and coronal 2D MRI series yielded 3D respiratory motion curves for all volunteers. The motion directionality and amplitude were very similar when measured directly as in-plane motion or estimated indirectly as through-plane motion. The mean peak-to-peak breathing amplitude was 1.6 mm (left-right), 11.0 mm (craniocaudal), and 2.5 mm (anterior-posterior). The position of the watermelon structure was estimated in 2D MRI images with a root-mean-square error of 0.52 mm (in-plane) and 0.87 mm (through-plane). Conclusions: A method for 3D tracking in 2D MRI series was developed and demonstrated for liver tracking in volunteers. The method would allow real-time 3D localization with integrated MR-Linac systems.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2014

Assessment of individual organ doses in a realistic human phantom from neutron and gamma stimulated spectroscopy of the breast and liver

Purpose: Understanding the radiation dose to a patient is essential when considering the use of an ionizing diagnostic imaging test for clinical diagnosis and screening. Using Monte Carlo simulations, the authors estimated the three-dimensional organ-dose distribution from neutron and gamma irradiation of the male liver, female liver, and female breasts for neutron- and gamma-stimulated spectroscopic imaging. Methods: Monte Carlo simulations were developed using the Geant4 GATE application and a voxelized XCAT human phantom. A male and a female whole body XCAT phantom was voxelized into 256 × 256 × 600 voxels (3.125 × 3.125 × 3.125 mm{sup 3}). A monoenergetic rectangular beam of 5.0 MeV neutrons or 7.0 MeV photons was made incident on a 2 cm thick slice of the phantom. The beam was rotated at eight different angles around the phantom ranging from 0° to 180°. Absorbed dose was calculated for each individual organ in the body and dose volume histograms were computed to analyze the absolute and relative doses in each organ. Results: The neutron irradiations of the liver showed the highest organ dose absorption in the liver, with appreciably lower doses in other proximal organs. The dose distribution within the irradiated slice exhibited substantial attenuation with increasing depth along the beam path, attenuating to ∼15% of the maximum value at the beam exit side. The gamma irradiation of the liver imparted the highest organ dose to the stomach wall. The dose distribution from the gammas showed a region of dose buildup at the beam entrance, followed by a relatively uniform dose distribution to all of the deep tissue structures, attenuating to ∼75% of the maximum value at the beam exit side. For the breast scans, both the neutron and gamma irradiation registered maximum organ doses in the breasts, with all other organs receiving less than 1% of the breast dose. Effective doses ranged from 0.22 to 0.37 mSv for the neutron scans and 41 to 66 mSv for the gamma scans. Conclusions: Neutron and gamma irradiation of a primary target organ was found to impart the majority of the total dose to the primary target organ (and other large organs) within the beam plane and considerably lower dose to proximal organs outside of the beam. These results also indicate that despite the use of a highly scattering particle such as a neutron, the dose from neutron stimulated emission computed tomography scans is on par with other clinical imaging techniques such as x-ray computed tomography (x-ray CT). Given the high nonuniformity in the dose across an organ during the neutron scan, care must be taken when computing average doses from neutron irradiations. The effective doses from neutron scanning were found to be comparable to x-ray CT. Further technique modifications are needed to reduce the effective dose levels from the gamma scans.

61 RADIATION PROTECTION AND DOSIMETRY↗
DOE OSTI2014

Poster — Thur Eve — 16: 4DCT simulation with synchronized contrast injection of liver SBRT patients

Stereotactic body radiation therapy (SBRT) has recently emerged as a valid option for treating liver metastases. SBRT delivers highly conformai dose over a small number of fractions. As such it is particularly sensitive to the accuracy of target volume delineation by the radiation oncologist. However, contouring liver metastases remains challenging for the following reasons. First, the liver usually undergoes significant motion due to respiration. Second, liver metastases are often nearly indistinguishable from the surrounding tissue when using computed tomography (CT) for imaging making it difficult to identify and delineate them. Both problems can be overcome by using four dimensional CT (4DCT) synchronized with intravenous contrast injection. We describe a novel CT simulation process which involves two 4DCT scans. The first scan captures the tumor and immediately surrounding tissue which in turn reduces the 4DCT scan time so that it can be optimally timed with intravenous contrast injection. The second 4DCT scan covers a larger volume and is used as the primary CT dataset for dose calculation, as well as patient setup verification on the treatment unit. The combination of two 4DCT scans, short and long, allows visualization of the liver metastases over all phases of breathing cycle while simultaneously acquiring long enough 4DCT dataset suitable for planning and patient setup verification.

60 APPLIED LIFE SCIENCES↗