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DOE OSTI2016

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells through targeting P4HA1 mRNA.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Possible roles of long-chain sphingomyelines and sphingomyelin synthase 2 in mouse macrophage inflammatory response

To evaluate the precise role of sphingomyelin synthase 2 (SMS2) in sphingomyelin (SM) metabolism and their anti-inflammatory properties, we analyzed species of major SM and ceramide (Cer) (18:1, 18:0 sphingoid backbone, C14 - C26 N-acyl part) in SMS2 knockout and wild-type mouse plasma and liver using HPLC-MS. SMS2 deficiency significantly decreased very long chain SM (SM (d18:1/22:0) and SM (d18:1/24:0 or d18:0/24:1)) and increased very long chain Cer (Cer (d18:1/24:0 or d18:0/24:1) and Cer (d18:1/24:1)), but not long chain SM (SM (d18:1/16:0), SM (d18:1/18:0 or d18:0/18:1) and SM (d18:1/18:1)) in plasma. To examine the effects of SM on inflammation, we studied the role of very long chain SM in macrophage activation. Addition of SM (d18:1/24:0) strongly upregulated several macrophage activation markers, SM (d18:1/6:0) and Cer (d18:1/24:0) however, did not. It was suggested that very long chain SM but not long chain SM were decreased in SMS2-deficient mice liver and plasma. And the exogenously added very long chain SM (d18:1/24:0) could activate macrophages directly, suggesting a novel role of plasma very long chain SM in modulating macrophage activation and resulting inflammation. - Highlights: • Very long-chain SM species were decreased in SMS2 knockout mouse plasma and liver. • Very long-chain ceramide species were increased in SMS2 knockout mouse plasma. • SMS2 deficiency diminished the inflammatory response of macrophages. • Very long-chain SM enhanced ICAM1 and iNOS expression in peritoneal macrophages.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Dipeptidyl peptidase-4 impairs insulin signaling and promotes lipid accumulation in hepatocytes

Dipeptidyl-peptidase 4 [DPP-4) has evolved into an important target in diabetes therapy due to its role in incretin hormone metabolism. In contrast to its systemic effects, cellular functions of membranous DPP-4 are less clear. Here we studied the role of DPP-4 in hepatic energy metabolism. In order to distinguish systemic from cellular effects we established a cell culture model of DPP-4 knockdown in human hepatoma cell line HepG2. DPP-4 suppression was associated with increased basal glycogen content due to enhanced insulin signaling as shown by increased phosphorylation of insulin-receptor substrate 1 (IRS-1), protein kinase B/Akt and mitogen-activated protein kinases (MAPK)/ERK, respectively. Additionally, glucose-6-phosphatase cDNA expression was significantly decreased in DPP-4 deficiency. Reduced triglyceride content in DPP-4 knockdown cells was paralleled by enhanced expressions of peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase −1 (CPT-1) while sterol regulatory element-binding protein 1c (SREBP-1c) expression was significantly decreased. Our data suggest that hepatic DPP-4 induces a selective pathway of insulin resistance with reduced glycogen storage, enhanced glucose output and increased lipid accumulation in the liver. Hepatic DPP-4 might be a novel target in fatty liver disease in patients with glucose intolerance. - Highlights: • DPP-IV knockdown results in increased insulin signaling in hepatocytes. • Increased fatty acid oxidation and decreased lipogenesis result in reduced hepatic triglyceride content in DPP-IV deficiency. • Hepatic DPP-IV induces a selective pathway of insulin resistance with increased triglyceride accumulation in the liver.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Down-regulation of TRAF4 targeting RSK4 inhibits proliferation, invasion and metastasis in breast cancer xenografts

Highlights: • Knockdown of TRAF4 inhibited the growth of xenograft tumors in nude mice. • Knockdown of TRAF4 significantly decreased the lung and liver metastases. • There is a negative correlation between TRAF4 and RSK4 mRNA and proteins. TRAF4 is interacting with RSK4 possibly by the induction of AKT/NF-κB signal axis. Ribosomal S6 protein kinase 4 (RSK4) was known as a novel tumor suppressor gene, and the tumor necrosis factor receptor-associated factor 4 (TRAF4) was linked to carcinogenesis. The purpose of this study is to further investigate the effect of the TRAF4 gene on cell proliferation, invasion and metastasis in vivo and explore whether there is an interaction between TRAF4 and RSK4 in breast cancer. MDA-MB-231 cells were transfected with lentivirus TRAF4-shRNA to specifically block the expression of TRAF4, or transfected with lentivirus negative-shRNA as a negative control. Four-six weeks female BALB/c nude mice were randomly assigned to three groups (n = 14): TRAF4-shRNA, negative and control, and then inoculated subcutaneously with the corresponding cells. In-vivo metastasis model was constructed by injecting above cells into tail vein. Tumor proliferation was assessed in terms of the tumor growth curve, tumor size and weight. Invasion and metastasis were evaluated by the histopathologic examination in lung or/and liver tissues. Measurement of TRAF4 and RSK4 expression and their correlation factors (AKT, NF-κB, TGF-β1, TNF-α, MMP2 and MMP9) were performed by immunohistochemistry, western blot or fluorescence quantitative RT-PCR. We found that the size and weight of tumors in TRAF4-shRNA group was significantly smaller than the negative and blank group, and the number of the lung and liver metastases lesions was also fewer (P < 0.05). And TRAF4 and its correlation factors (P-AKT, P-NF-κB, TGF-β1, TNF-α, MMP2 and MMP9) in the TRAF4-shRNA group were significantly decreased compared with the negative and blank group. However, the expression of RSK4 mRNA and protein in TRAF4-shRNA group were significantly increased. Collectively, TRAF4 knockdown significantly inhibited proliferation, invasion and metastasis in the xenograft nude mouse model, possibly involving in the interaction with RSK4 through down-regulation of AKT signaling pathway and then inactivating NF-κB pathway.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Hepatocyte-specific deletion of Brg1 alleviates methionine-and-choline-deficient diet (MCD) induced non-alcoholic steatohepatitis in mice

Highlights: • Brg1 expression is up-regulated by pro-NASH stimuli in vitro and in vivo. • Hepatocyte-specific Brg1 deletion alleviates non-alcoholic steatohepatitis in mice. • Brg1 is essential for the interaction between MRTF-A and NF-κB. • Brg1 promotes the activation of SREBP1c target genes in mice. Uncontrolled inflammatory response and augmented lipid accumulation represent two key pathophysiological events in the pathogenesis of non-alcoholic steatohepatitis (NASH). NF-κB and SREBP1c program transcriptional regulation of cellular inflammatory response and lipid metabolism, respectively. The epigenetic mechanism underlying NF-κB-dependent pro-inflammatory transcription and SREBP1c-dependent pro-lipogenic transcription remains incompletely understood. In the present study we investigated the involvement of Brg1, a chromatin remodeling protein, in NASH pathogenesis in a methionine-and-choline deficient diet (MCD) induced mouse model. Brg1 expression was up-regulated in the liver in mice fed on the MCD diet and in primary hepatocytes exposed to free fatty acids. Liver injury and hepatic inflammation attenuated in hepatocyte-specific Brg1 knockout (CKO) mice fed on the MCD diet compared to the wild type (WT) littermates. Likewise, synthesis of pro-inflammatory mediators was down-regulated in primary hepatocytes isolated from CKO mice compared to WT mice, which resulted in reduced macrophage chemotaxis. Brg1 contributed to the transcription of pro-inflammatory mediators possibly by regulating the interaction between NF-κB and its co-factor MRTF-A. On the other hand, accumulation of triglyceride and cholesterol was ameliorated in MCD-fed CKO mice with a concomitant reduction of SREBP1c target genes. Brg1 interacted with SREBP1c and modulated the transcription of SREB1c target genes in the liver in response to MCD feeding by influencing active histone modifications. In conclusion, targeting Brg1 may yield novel anti-NASH therapeutics by simultaneously normalizing hepatic inflammatory status and metabolic profile in NASH patients.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Epithelial cell adhesion molecule expression in hepatic stem/progenitor cells is controlled by the molecular clock system

Highlights: • We investigated clock gene effects on EpCAM-positive cell induction in hepatitis. • Hepatitis in CLOCK mutant mice was severe compared with wild-type mice. • CLOCK regulated expression of LEF, which regulated EpCAM expression. • CLOCK was important for healing hepatitis. The circadian rhythm, which regulates various body functions, is transcriptionally controlled by a series of clock gene clusters. The clock genes are related to the pathology of various kinds of diseases. Although there is evidence of serious sleep disorders in patients with chronic hepatitis, the liver regeneration mechanism under chronic hepatitis conditions and its association with the clock genes are not clear. In this study, the influence of the circadian locomotor output cycles kaput (CLOCK), which is one of the clock genes, on a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced hepatitis animal model was investigated. The appearance of potential hepatic stem-like cells (epithelial cell adhesion molecule [EpCAM]-positive cells) is an initial critical step in liver regeneration during chronic inflammation. The results showed a considerable number of hepatic EpCAM-positive cells in the wild-type (WT) mice 1 week after the DDC feeding. However, the number of EpCAM-positive cells in the Clock-mutant (Clk/Clk) mice decreased, and their hepatitis was worse compared with the WT mice. In addition, the expression of Epcam mRNA, which is a functional marker of potential hepatic stem-like cells, was controlled by LEF1, which was regulated by CLOCK. The results of this study will facilitate the elucidation of the liver regeneration mechanisms, including those at the molecular level, and may assist in the development of new treatment modalities in the future.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Immunological responses against hepatitis B virus in human peripheral blood mononuclear cell-engrafted mice

Highlights: • DKO-NOG mice, which were NOG mice deficient in both MHC class I and II, were established. • Liver damage was reduced, and survival time was prolonged in human PBMC-engrafted DKO-NOG mice. • HBV-specific immune responses were observed in the established DKO-NOG mice. It is well known that immune-mediated virus elimination is necessary for the treatment of HBV infection. Reconstitution of human immune cells in liver chimeric mice is warranted to understand the immunopathogenesis of HBV infection. Here, we report a new immunologically humanized mouse model with a human immune system via reconstitution of immunodeficient NOG-Iaβ/β2 m double KO mice, which are NOG mice that are deficient in both MHC class I and II (DKO-NOG mice), with human HLA-A2-positive peripheral blood mononuclear cells (PBMCs). After injection of PBMCs, the xenogeneic graft-versus-host disease observed in PBMC-engrafted NOG mice was prevented in PBMC-engrafted DKO-NOG mice. Liver damage was reduced, and the survival time was prolonged in human PBMC-engrafted DKO-NOG mice compared to those in the NOG mice. The expression levels of PD-1 and Tim-3 on human T cells from PBMC-engrafted DKO-NOG mice were lower than those from NOG mice. By induction of HBV-specific T cell responses, such as vaccination with HBc-derived, peptide-pulsed DCs, hydrodynamic injection of HBV vector and intrasplenic injection of HepG2.2.15, the number of HBc-derived, peptide-specific CTLs increased in PBMC-engrafted DKO-NOG mice. Moreover, the recombinant HBV vaccine resulted in the production of hepatitis B surface antibody in 50% of the vaccinated mice. The induction of HBV-specific immune responses could be established in the immunologically humanized mice.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Deletion of hephaestin and ceruloplasmin induces a serious systemic iron deficiency and disrupts iron homeostasis

Highlights: • Heph/Cp double knockout mice. • Systemic iron deficiency. • Local tissue iron overload. Multi-copper ferroxidases (MCFs) play important roles in cellular iron metabolism and homeostasis. In this study, we generated the hephaestin (Heph), ceruloplasmin (Cp) single and Heph/Cp double knockout (KO) mice to investigate the roles of MCFs in iron transport among system and vital organs in mice at 4 weeks and 6 months of age. Compared with wild-type (WT) mice, Heph/Cp mice at both ages presented with severe anemia and significantly lower iron level in the serum and spleen, but with significantly higher iron level in the liver, heart, kidney, and duodenal enterocytes. Furthermore, Heph/Cp mice displayed significantly lower level of hepcidin mRNA and transferrin receptor 1 (TFR1) protein expression, but significantly higher level of ferroportin 1 (FPN1) protein expression in the liver than WT mice at 6 months of age. Liver superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities were significantly lower in Heph/Cp KO mice than WT mice at 6 months of age. Together, our results suggest that ablation of HEPH and CP could lead to severe systemic iron deficiency and local tissue iron overload, which disrupt the whole body iron homeostasis and impact on tissue functions.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Low doses of multi-walled carbon nanotubes elicit hepatotoxicity in rats with markers of oxidative stress and induction of pro-inflammatory cytokines

The investigation into the potential health risks associated with the use of engineered nanoparticles is a major scientific interest in recent years. The present study elucidated the involvement of pro-inflammatory cytokines, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in carboxylated multi-walled carbon nanotubes (MWCNTs)-induced hepatotoxicity. Pubertal rats were exposed to purified MWCNTs at 0, 0.25, 0.50, 0.75 and 1.0 mg/kg for 5 consecutive days. Results indicated that exposure to MWCNTs caused liver damage evidenced by significant elevation in serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) when compared with control. Moreover, MWCNTs significantly decreased superoxide dismutase (SOD) and glutathione S-transferase (GST) activities as well as glutathione level whereas it significantly increased catalase (CAT) and glutathione peroxidase (GPx) activities in liver of the treated rats. Moreover, the dose-dependent increase in hepatic hydrogen peroxide (H{sub 2}O{sub 2}) and lipid peroxidation levels were accompanied by marked increase in micronucleated polychromatic erythrocytes (MNPCE) in the MWCNTs-treated rats. Administration of MWCNTs significantly increased serum concentrations of pro-inflammatory cytokines namely interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the treated rats. Immunohistochemical analysis showed significantly increased COX-2 and iNOS protein expressions in the liver of MWCNTs-treated rats. In conclusion, carboxylated MWCNTs induces hepatic damage via disruption of antioxidant defense systems, promotion of pro-inflammatory cytokines generation and expression of COX-2 and i-NOS in rats.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Identification of biologically active δ-lactone eicosanoids as paraoxonase substrates

Highlights: • Native substrates for the PON family of esterases are uncertain. • PON3 efficiently hydrolyzed two bioactive eicosanoid δ-lactones. • PONs were the predominant enzymes contributing to hydrolysis of the δ-lactones in mouse and human liver lysates. • Endogenous bioactive polyunsaturated fatty acid lactones may be a class of native PON substrates. The mammalian paraoxonases (PONs 1, 2 and 3) are a family of esterases that are highly conserved within and between species. They exhibit antioxidant and anti-inflammatory activities. However, their physiological function(s) and native substrates are uncertain. Previous structure-activity relationship studies demonstrate that PONs have a high specificity for lipophilic lactones, suggesting that such compounds may be representative of native substrates. This report describes the ability of PONs to hydrolyze two bioactive δ-lactones derived from arachidonic acid, 5,6-dihydroxy-eicosatrienoic acid lactone (5,6-DHTL) and cyclo-epoxycyclopentenone (cyclo-EC). Both lactones were very efficiently hydrolyzed by purified PON3. PON1 efficiently hydrolyzed 5,6-DHTL, but with a specific activity about 15-fold lower than PON3. 5,6-DHTL was a poor substrate for PON2. Cyclo-EC was a poor substrate for PON1 and not hydrolyzed by PON2. Studies with the PON inhibitor EDTA and a serine esterase inhibitor indicated that the PONs are the main contributors to hydrolysis of the lactones in human and mouse liver homogenates. Studies with homogenates from PON3 knockout mouse livers indicated that >80% of the 5,6-DHTL and cyclo-EC lactonase activities were attributed to PON3. The findings provide further insight into the structural requirements for PONs substrates and support the hypothesis that PONs, particularly PON1 and PON3, evolved to hydrolyze and regulate a class of lactone lipid mediators derived from polyunsaturated fatty acids.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Retinoic acid modulates iron metabolism imbalance in anemia of inflammation induced by LPS via reversely regulating hepcidin and ferroportin expression

Highlights: • Anemia of inflammation (AI) model was induced by lipopolysaccharide in mouse. • Retinoic acid (RA) increases RBC, Hb, HCT, MCV, MCHC and EPO in AI model. • RA inhibits inflammation in AI model by decreasing TNF-α, IL-18 and IL-1β contents. • RA modulates iron metabolism imbalance in AI model. • RA reversely regulates hepcidin and ferroportin expression in AI model. The present study was designed to investigate the effect of retinoic acid (RA) on anemia of inflammation (AI) induced by lipopolysaccharide (LPS) and explore the potential mechanisms. BALB/c mice were randomly assigned into four groups: control group; LPS (10 mg/kg) group, LPS + RA (3 mg/kg) and LPS + RA (15 mg/kg) groups. Red blood cell count (RBC), hemoglobulin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin contentration (MCHC), erythropoietin (EPO) and iron content in both serum and liver tissue were measured. The AI model induced by LPS was successfully established represented by the decreases in RBC, Hb, HCT, MCV, MCHC and EPO for anemia indicators and by the increases in TNF-α, IL-18 and IL-1β contents for inflammation indicators. However, supplementation of RA increased the levels of anemia indicators and decreased the content of inflammation indicators. In addition, RA increased the content of iron in serum, while decreased its content in liver tissue. Furthermore, RA down-regulated the protein expression of hepcidin, toll-like receptor 4 (TLR4) and p-p65 in liver tissue, while up-regulated that of ferroportin. RA modulates iron metabolism imbalance in AI induced by LPS via reversely regulating hepcidin and ferroportin expression, which might be mediated by TLT-4/NFκB signaling pathway.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2013

Urinary arsenic profiles and the risks of cancer mortality: A population-based 20-year follow-up study in arseniasis-endemic areas in Taiwan

Few studies investigated the association between chronic arsenic exposure and the mortality of cancers by estimating individual urinary arsenic methylation profiles. Therefore, we compared with the general population in Taiwan to calculate the standardized mortality ratio (SMR) in arseniasis-endemic area of Taiwan from 1996 to 2010 and evaluated the dose-response relationships between environmental arsenic exposure indices or urinary arsenic profiles and the mortality of cause-specific cancer. A cohort of 1563 residents was conducted and collected their urine sample and information regarding arsenic exposure from a questionnaire. All-cause death was identified using the National Death Registry of Taiwan. Urinary arsenic profiles were measured using high performance liquid chromatography–hydride generator–atomic absorption spectrometry. We used Cox proportional hazard models to evaluate the mortality risks. In results, 193 all-site cancer deaths, and 29, 71, 43 deaths respectively for liver, lung and bladder cancers were ascertained. The SMRs were significantly high in arseniasis-endemic areas for liver, lung, and bladder cancers. People with high urinary InAs% or low DMA% or low secondary methylation index (SMI) were the most likely to suffer bladder cancer after adjusting other risk factors. Even stopping exposure to arsenic from the artesian well water, the mortality rates of the residents were higher than general population. Finally, urinary InAs%, DMA% and SMI could be the potential biomarkers to predict the mortality risk of bladder cancer. -- Highlights: ► The SMRs were significantly high in arseniasis-endemic areas for liver, lung, and bladder cancers. ► People with high urinary InAs% were the most likely to suffer bladder cancer. ► People with low DMA% or low SMI were the most likely to suffer bladder cancer.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2014

Metals in tissues of migrant semipalmated sandpipers (Calidris pusilla) from Delaware Bay, New Jersey

There is an abundance of field data on levels of metals for feathers in a variety of birds, but relatively few data for tissues, especially for migrant species from one location. In this paper we examine the levels of arsenic, cadmium, chromium, lead, manganese, mercury and selenium in muscle, liver, brain, fat and breast feathers from migrant semipalmated sandpipers (Calidris pusilla) collected from Delaware Bay, New Jersey. Our primary objectives were to (1) examine variation as a function of tissue, (2) determine the relationship of metal levels among tissues, and (3) determine the selenium:mercury molar ratio in different tissues since selenium is thought to protect against mercury toxicity. We were also interested in whether the large physiological changes that occur while shorebirds are on Delaware Bay (e.g. large weight gains in 2–3 weeks) affected metal levels, especially in the brain. There were significant differences among tissues for all metals. The brain had the lowest levels of arsenic and cadmium, and was tied for the lowest levels of all other metals except lead and selenium. Correlations among metals in tissues were varied, with mercury levels being positively correlated for muscle and brain, and for liver and breast feathers. Weights vary among individuals at the Delaware Bay stopover, as they arrive light, and gain weight prior to migration north. Bird weight and levels of arsenic, cadmium, and selenium in the brain were negatively correlated, while they were positively correlated for lead. There was no positive correlation for mercury in the brain as a function of body weight. The selenium:mercury molar ratio varied significantly among tissues, with brain (ratio of 141) and fat having the highest ratios, and liver and breast feathers having the lowest. In all cases, the ratio was above 21, suggesting the potential for amelioration of mercury toxicity. - Highlights: • Metal levels were examined for migrant semipalmated sandpipers. • There were differences in metal levels among internal tissues. • Brain had the lowest levels of arsenic and cadmium. • Bird weight and arsenic, cadmium, and selenium levels in brain were negatively correlated. • Selenium:mercury molar ratio varied among tissues (21–141, suggesting protection)

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2014

A systematic study of the disposition and metabolism of mercury species in mice after exposure to low levels of thimerosal (ethylmercury)

Thimerosal (TM) is an ethylmercury (etHg)-containing preservative used in some vaccines despite very limited knowledge on the kinetics and direct interaction/effects in mammals' tissues after exposure. Thus, this study aimed to evaluate the kinetics of Hg species in mice in a time course analysis after intramuscular injection of TM, by estimating Hg half-lives in blood and tissues. Mice were exposed to one single intramuscular dose of 20 µg of Hg as TM. Blood, brain, heart, kidney and liver were collected at 0.5 hour (h), 1 h, 8 h, 16 h, 144 h, 720 h and 1980 h after TM exposure (n=4). Hg species in animal tissues were identified and quantified by speciation analysis via liquid chromatography hyphenated with inductively coupled mass spectrometry (LC–ICP-MS). It was found that the transport of etHg from muscle to tissues and its conversion to inorganic Hg (inoHg) occur rapidly. Moreover, the conversion extent is modulated in part by the partitioning between EtHg in plasma and in whole blood, since etHg is rapidly converted in red cells but not in a plasma compartment. Furthermore, the dealkylation mechanism in red cells appears to be mediated by the Fenton reaction (hydroxyl radical formation). Interestingly, after 0.5 h of TM exposure, the highest levels of both etHg and inoHg were found in kidneys (accounting for more than 70% of the total Hg in the animal body), whereas the brain contributed least to the Hg body burden (accounts for <1.0% of total body Hg). Thirty days after TM exposure, most Hg had been excreted while the liver presented the majority of the remaining Hg. Estimated half-lives (in days) were 8.8 for blood, 10.7 for brain, 7.8 for heart, 7.7 for liver and 45.2 for kidney. Taken together, our findings demonstrated that TM (etHg) kinetics more closely approximates Hg{sup 2+} than methylmercury (meHg) while the kidney must be considered a potential target for etHg toxicity. - Highlights: • Ethylmercury is rapidly converted to inorganic mercury. • Hg substantially accumulates in kidney with a terminal half-life of 45.2 d. • The dealkylation of ethylmercury occurs in red blood cells but not in plasma. • Hydroxyl radical is probably the main effector of this dealkylation. • Kidney must be considered a potential target for ethylmercury toxicity.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Are ingested plastics a vector of PCB contamination in northern fulmars from coastal Newfoundland and Labrador?

Highlights: • The ingestion of plastics can lead to increased contaminant exposure. • We examined PCB concentrations in seabirds and their ingested plastics. • No significant correlations were found between PCBs and ingest plastics. • There were differences in the PCB concentrations and congener profiles in plastics and livers. • Xenobiotics, such as PCBs, may undergo metabolism in upper trophic level species. While marine animals are exposed to environmental contaminants via their prey, because plastic pollution in the aquatic environment can concentrate some chemicals, ingested plastics are thought to increase the exposure of biota to contaminants. Currently, in the literature there are contradictory results relating to how higher levels of ingested plastics by birds may lead to higher levels of polychlorinated biphenyl (PCBs). To date none of these have incorporated known Toxic Equivalency Factors (TEFs) for non-ortho and mono-ortho congeners of PCB which is critical to assessing the potential effects from PCBs. We examined northern fulmars (Fulmarus glacialis) from the Labrador Sea region Canada, and the ingested plastics from these same birds for comparative PCB concentrations. We found no significant correlations between the PCB concentrations in the birds and the mass or number of retained ingested plastic pieces in the stomach, this held true when PCBs were considered by a number of different ways, including ∑{sub 4}PCB, ∑PCB, lower-chlorinated, high-chlorinated, non-ortho PCB, and mono-ortho congeners. PCB concentrations were lower in plastics as compared with livers. We found significant differences in congener profiles between the ingested plastics and seabird livers suggesting that while plastics do not contribute to the PCB concentrations, there may be some interactions between plastics and the chemicals that the birds are exposed to via ingested plastics.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2011

Intensity-Modulated Proton Therapy Further Reduces Normal Tissue Exposure During Definitive Therapy for Locally Advanced Distal Esophageal Tumors: A Dosimetric Study

Purpose: We have previously found that {<=} 75% of treatment failures after chemoradiotherapy for unresectable esophageal cancer appear within the gross tumor volume and that intensity-modulated (photon) radiotherapy (IMRT) might allow dose escalation to the tumor without increasing normal tissue toxicity. Proton therapy might allow additional dose escalation, with even lower normal tissue toxicity. In the present study, we compared the dosimetric parameters for photon IMRT with that for intensity-modulated proton therapy (IMPT) for unresectable, locally advanced, distal esophageal cancer. Patients and Methods: Four plans were created for each of 10 patients. IMPT was delivered using anteroposterior (AP)/posteroanterior beams, left posterior oblique/right posterior oblique (LPO/RPO) beams, or AP/LPO/RPO beams. IMRT was delivered with a concomitant boost to the gross tumor volume. The dose was 65.8 Gy to the gross tumor volume and 50.4 Gy to the planning target volume in 28 fractions. Results: Relative to IMRT, the IMPT (AP/posteroanterior) plan led to considerable reductions in the mean lung dose (3.18 vs. 8.27 Gy, p < .0001) and the percentage of lung volume receiving 5, 10, and 20 Gy (p {<=} .0006) but did not reduce the cardiac dose. The IMPT LPO/RPO plan also reduced the mean lung dose (4.9 Gy vs. 8.2 Gy, p < .001), the heart dose (mean cardiac dose and percentage of the cardiac volume receiving 10, 20, and 30 Gy, p {<=} .02), and the liver dose (mean hepatic dose 5 Gy vs. 14.9 Gy, p < .0001). The IMPT AP/LPO/RPO plan led to considerable reductions in the dose to the lung (p {<=} .005), heart (p {<=} .003), and liver (p {<=} .04). Conclusions: Compared with IMRT, IMPT for distal esophageal cancer lowered the dose to the heart, lung, and liver. The AP/LPO/RPO beam arrangement was optimal for sparing all three organs. The dosimetric benefits of protons will need to be tailored to each patient according to their specific cardiac and pulmonary risks. IMPT for esophageal cancer will soon be investigated further in a prospective trial at our institution.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

DOE OSTI2012

Stereotactic Hypofractionated Radiation Therapy as a Bridge to Transplantation for Hepatocellular Carcinoma: Clinical Outcome and Pathologic Correlation

Purpose: We sought to determine efficacy, safety, and outcome of stereotactic hypofractionated radiation therapy (SHORT) as a suitable bridging therapy for patients awaiting liver transplantation (LT) for hepatocellular carcinoma (HCC). We also examined histological response to radiation in the resected or explanted livers. Methods and Materials: Between August 2007 and January 2009, 18 patients with 21 lesions received SHORT. A median total dose of 50 Gy was delivered in 10 fractions. Three patients underwent either chemoembolization (n = 1) or radiofrequency ablation (n = 2) prior to SHORT. Radiographic response was based on computed tomography evaluation at 3 months after SHORT. Histological response as a percentage of tumor necrosis was assessed by a quantitative morphometric method. Results: Six of 18 patients were delisted because of progression (n = 3) or other causes (n = 3). Twelve patients successfully underwent major hepatic resection (n = 1) or LT (n = 11) at a median follow-up of 6.3 months (range, 0.6-11.6 months) after completion of SHORT. No patient developed gastrointestinal toxicity Grade {>=}3 or radiation-induced liver disease. Ten patients with 11 lesions were evaluable for pathological response. Two lesions had 100% necrosis, three lesions had {>=}50% necrosis, four lesions had {<=}50% necrosis, and two lesions had no necrosis. All patients were alive after LT and/or major hepatic resection at a median follow-up of 19.6 months. Conclusions: SHORT is an effective bridging therapy for patients awaiting LT for HCC. It provides excellent in-field control with minimal side effects, helps to downsize or stabilize tumors prior to LT, and achieves good pathological response.

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