Faculty Bibliography

The concentration of UV filters (UVFs) and UV light stabilizers (UVLSs) were measured in seawater and river water collected from sites at four beaches, two reefs, and one river on Okinawa Island, Japan. UVFs and/or UVLSs of 8-10 types were detected in beaches samples and 6-9 types were detected in reef samples. The total UVF concentrations at the beach sites were highest either in July or August with a maximum of 1.4 mug L(-1). The concentrations at the reef sites did not show peaks in summer and the maximum values were close to 10 ng L(-1). The detected UVF profiles reflected the ingredients of sunscreens used in each region. The highest UVLS concentrations at the reefs were observed not only in summer but also in June and September. The UVLS concentrations at the reefs were similar to or even higher than that at the beaches or in the river.

Residue levels and patterns of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), their hydroxylated metabolites (OH-PCBs, OH-PBDEs), and methoxylated PBDEs (MeO-PBDEs) in the blood of various terrestrial mammals in Japan, including cats, raccoon dogs, dogs, masked palm civets, foxes, raccoons, badgers, and mongooses were determined. Tri- through penta-chlorinated OH-PCBs were predominant in cat blood, whereas hexa- through octa-chlorinated OH-PCBs were found in other species. High proportion of BDE209 was found in all species, suggesting exposure to municipal waste and soil containing higher levels of deca-BDE products. 6OH-/MeO-BDE47 and 2'OH-/MeO-BDE68 were dominant in all terrestrial mammals. This is first report on the detection of OH-/MeO-PBDEs in the blood of terrestrial mammals. High concentrations of OH-/MeO-PBDEs were found in cats, suggesting the intake of these compounds from seafood. Cats exhibited higher accumulation and specific patterns of OH-PCBs, OH-PBDEs, and MeO-PBDEs, they may be at a high risk from these metabolites.

The INrf2 (Keap1)-Nrf2 cell sensor complex has a crucial role in protection against chemical- and radiation-induced oxidative stress and cellular transformation. INrf2, in association with Cul3-Rbx1, ubiquitylates and degrades Nrf2. Exposure to stressors leads to stabilization of Nrf2 and the coordinated activation of cytoprotective proteins and cellular protection. However, the molecular signal(s) that regulate control of Nrf2 by INrf2 remain elusive. In this report, we demonstrate that phosphorylation of INrf2 at Ser599 and Ser602 by the oncoprotein PKCepsilon is essential for INrf2-Nrf2 interaction, and the subsequent ubiquitylation and degradation of Nrf2. Inhibition of PKCepsilon, knockdown of PKCepsilon and the INrf2S602A mutant all failed to phosphorylate INrf2, leading to loss of the INrf2-Nrf2 interaction, Nrf2 degradation and enhanced cytoprotection and drug resistance. Molecular modeling analyses revealed that phosphorylation of S599 exposes the deeply buried S602 for phosphorylation and enhanced INrf2-Nrf2 interaction. Analysis of human lung and liver tumor protein arrays showed lower PKCepsilon and higher Nrf2 levels, which presumably promoted cancer cell survival and drug resistance. In conclusion, phosphorylation of INrf2 by PKCepsilon leads to regulation of Nrf2, with significant implications for the survival of cancer cells, which often express lower levels of PKCepsilon.

NQO1 [NAD(P)H quinone oxidoreductase 1; also known as DT-diaphorase] is a cytosolic enzyme that catalyses the two-electron reduction of various quinones including vitamin K. The enzyme may play a role in vitamin K metabolism by reducing vitamin K to vitamin K hydroquinone for utilization in the post-translational gamma-glutamyl carboxylation reactions required by several proteins involved in blood coagulation. The aim of the present study was to assess the contribution of NQO1 to vitamin K reduction and haemostasis in an in vivo model. We examined the contribution of NQO1 to haemostasis by examining survival rates in mice poisoned with the anticoagulant warfarin. Supraphysiological amounts of vitamin K sufficiently reversed the effects of warfarin in both wild-type and NQO1-deficient mice. Additionally, vitamin K reductase activities distinct from VKOR (vitamin K epoxide reductase) and NQO1 were measured in vitro from both wild-type and NQO1-defecient mice. The results of the present study suggest that NQO1 does not play a major role in the production of vitamin K hydroquinone and supports the existence of multiple vitamin K reduction pathways. The properties of a NAD(P)H-dependent vitamin K reductase different from NQO1 are described.

The accessible treatment options for life-threatening neglected visceral leishmaniasis (VL) disease have problems with efficacy, stability, adverse effects, and cost, making treatment a complex issue. Here we formulated nanometric amphotericin B (AmB)-encapsulated chitosan nanocapsules (CNC-AmB) using a polymer deposition technique mediated by nanoemulsion template fabrication. CNC-AmB exhibited good steric stability in vitro, where the chitosan content was found to be efficient at preventing destabilization in the presence of protein and Ca(2+). A toxicity study on the model cell line J774A and erythrocytes revealed that CNC-AmB was less toxic than commercialized AmB formulations such as Fungizone and AmBisome. The results of in vitro (macrophage-amastigote system; 50% inhibitory concentration [IC(50)], 0.19 +/- 0.04 mug AmB/ml) and in vivo (Leishmania donovani-infected hamsters; 86.1% +/- 2.08% parasite inhibition) experiments in conjunction with effective internalization by macrophages illustrated the efficacy of CNC-AmB at augmenting antileishmanial properties. Quantitative mRNA analysis by real-time PCR (RT-PCR) showed that the improved effect was synergized with the upregulation of tumor necrosis factor alpha (TNF-alpha), interleukin-12 (IL-12), and inducible nitric oxide synthase and with the downregulation of transforming growth factor beta (TGF-beta), IL-10, and IL-4. These research findings suggest that a cost-effective CNC-AmB immunoadjuvant chemotherapeutic delivery system could be a viable alternative to the current high-cost commercial lipid-based formulations.

Angiogenesis is mediated by signaling through receptor tyrosine kinases (RTKs), Src family kinases and adhesion receptors such as integrins, yet the mechanism how these signaling pathways regulate one another remains incompletely understood. The RTK modulator, Sprouty4 (Spry4) inhibits endothelial cell functions and angiogenesis, but the mechanisms remain to be fully elucidated. In this study, we demonstrate that Spry4 regulates angiogenesis in part by regulating endothelial cell migration. Overexpression of Spry4 in human endothelial cells inhibited migration and adhesion on vitronectin (VTN), whereas knockdown of Spry4 enhanced these behaviors. These activities were shown to be c-Src-dependent and Ras-independent. Spry4 disrupted the crosstalk between vascular endothelial growth factor-2 and integrin alphaVbeta3, the receptor for VTN. Spry4 overexpression resulted in decreased integrin beta3 protein levels in a post-transcriptional manner in part by modulating its tyrosine phosphorylation by c-Src. Conversely, knockdown of Spry4 resulted in increased integrin beta3 protein levels and tyrosine phosphorylation. Moreover, in vivo analysis revealed that Spry4 regulated integrin beta3 levels in murine embryos and yolk sacs. Our findings identify an unanticipated role for Spry4 in regulating c-Src activity and integrin beta3 protein levels, which contributes to the regulation of migration and adhesion of endothelial cells. Thus, targeting Spry4 may be exploited as a target in anti-angiogenesis therapies.

Nuclear transcription factor Nrf2 binds with the antioxidant-response element (ARE) in the promoter regions of cytoprotective genes, leading to their increased expression and cellular protection. In this study, we investigated the role of Nrf2 in the regulation of antiapoptotic Bcl-xL protein and its effect on cellular apoptosis. Treatment of mouse Hepa-1 cells with the antioxidant tert-butylhydroquinone led to the induction of Bcl-xL gene expression. Promoter mutagenesis, transfection, and chromatin immunoprecipitation assays identified an ARE between nucleotides -608 and -600 in the forward strand of the proximal Bcl-xL promoter that bound to Nrf2 and led to increased Bcl-xL gene expression. In addition, short interfering RNA (siRNA) inhibition and overexpression of Nrf2 led to a respective decrease and increase in Bcl-xL gene expression. These results implicated Nrf2 in the regulation of expression and induction of Bcl-xL protein. Nrf2-mediated expression of Bcl-xL protein downregulated Bax and decreased caspase 3/7 activity. SiRNA inhibition of both Nrf2 and Bcl-xL increased the susceptibility of cancer cells to etoposide-mediated cell death and reduced cell survival. Moreover, dysfunctional/mutant INrf2 (inhibitor of Nrf2) in human lung cancer cells failed to degrade Nrf2, resulting in increased Bcl-xL levels and increased cell survival. These data provide the first evidence of Nrf2 in the control of Bcl-xL expression and apoptotic cell death with implications for antioxidant protection, survival of cancer cells, and drug resistance.

Experimental animals offered continuous 24-hour free choice access to ethanol rarely display voluntary ethanol consumption at levels sufficient to induce intoxication or to engender dependence. One of the simplest ways to increase voluntary ethanol intake is to impose temporal limitations on ethanol availability. Escalation of ethanol intake has been observed in both rats and mice under a variety of different schedules of alternating ethanol access and deprivation. Although such effects have been observed in a variety of rat and mouse genotypes, little is known concerning possible genetic correlations between responses to intermittent ethanol access and other ethanol-related phenotypes. In the present study, we examined the effects of intermittent ethanol access in mouse genotypes characterized by divergent responses to ethanol in other domains, including ethanol preference (C57BL/6J and C3H/HeJ mice), binge-like ethanol drinking (High Drinking in the Dark and HS/Npt mice) and ethanol withdrawal severity (Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice). Although intermittent ethanol access resulted in escalated ethanol intake in all tested genotypes, the robustness of the effect varied across genotypes. On the other hand, we saw no evidence that the effects of intermittent access are correlated with either binge-like drinking or withdrawal severity, and only weak evidence for a genetic correlation with baseline ethanol preference. Thus, these different ethanol-related traits appear to depend on largely unique sets of genetic mediators.