Bioaccumulation and primary risk assessment of persistent organic pollutants with various bivalves.

Field surveys on persistent organic pollutant (POP) bioaccumulation were conducted with oysters, clams and scallops whose consumption amount accounted for large shares in the total consumption of shellfish in Japan. There was no numerical difference in bioaccumulation characteristics between oysters, clams, scallops, Corbicula and Mytilus galloprovincialis. Therefore, it was clear that the bioaccumulation characteristics in oysters, clams and scallops, which are important for food, could be ascertained by using the monitoring results with Corbicula and M. galloprovincialis which are easily sampled in various water areas in the world. Non-cancer risk (hazard quotient, HQ) and cancer risk (excess cancer risk, ΔR) via shellfish ranged from 10⁻8 to 10⁻4 and from 10⁻¹¹ to 10⁻7, respectively, at sampling points, which showed the risks of POP exposure via shellfish to be low enough. However, concerning the intake of other food, the importance of dieldrin monitoring should be suggested in Japan. Based on these results, the effectiveness of primary risk assessment could be suggested for screening chemicals whose preferential monitoring is needed.

Hydroxylation and dechlorination of 3,3',4,4'-tetrachlorobiphenyl (CB77) by rat and human CYP1A1s and critical roles of amino acids composing their substrate-binding cavity.

Cytochrome P450 (CYP) monooxygenases play critical roles in determining the toxicity of polychlorinated biphenyls (PCBs) in mammals. Hydroxylation of PCBs by these enzymes leads to increased water solubility, promoting the elimination of PCBs from the body. The CYP1 family is mainly responsible for metabolizing PCBs that exhibit a dioxin-like toxicity. Although the dioxin-like PCB 3,3',4,4'-tetrachlorobiphenyl (CB77) is abundant in the environment and accumulates in organisms, information on CB77 metabolism by CYP1A1s is limited. In this study, recombinant rat CYP1A1 metabolized CB77 to 4'-hydroxy (OH)-3,3',4,5'-tetrachlorobiphenyl (CB79) and 4'-OH-3,3',4-trichlorobiphenyl (CB35), whereas human CYP1A1 produced only 4'-OH-CB79. Rat CYP1A1 exhibited much higher metabolizing activity than human CYP1A1 because CB77 was stably accommodated in the substrate-binding cavity of rat CYP1A1 and was close to its heme. In a rat CYP1A1 mutant with two human-type amino acids, the production of 4'-OH-CB79 decreased, whereas that of the dechlorinated metabolite 4'-OH-CB35 increased. These results are explained by a shift in the CB77 positions toward the heme. This study provides insight into the development of enzymes with high metabolizing activity and clarifies the structural basis of PCB metabolism, as dechlorination contributes to a drastic decrease in dioxin-like toxicity.

Cytokine-dependent modification of IL-12p70 and IL-23 balance in dendritic cells by ligand activation of Valpha24 invariant NKT cells.

CD1d-restricted invariant NKT (iNKT) cells play crucial roles in various types of immune responses, including autoimmune diseases, infectious diseases and tumor surveillance. The mechanisms underlying their adjuvant functions are well understood. Nevertheless, although IL-4 and IL-10 production characterize iNKT cells able to prevent or ameliorate some autoimmune diseases and inflammatory conditions, the precise mechanisms by which iNKT cells exert immune regulatory function remain elusive. This study demonstrates that the activation of human iNKT cells by their specific ligand alpha-galactosylceramide enhances IL-12p70 while inhibiting the IL-23 production by monocyte-derived dendritic cells, and in turn down-regulating the IL-17 production by memory CD4(+) Th cells. The ability of the iNKT cells to regulate the differential production of IL-12p70/IL-23 is mainly mediated by a remarkable hallmark of their function to produce both Th1 and Th2 cytokines. In particular, the down-regulation of IL-23 is markedly associated with a production of IL-4 and IL-10 from iNKT cells. Moreover, Th2 cytokines, such as IL-4 and IL-13 play a crucial role in defining the biased production of IL-12p70/IL-23 by enhancement of IL-12p70 in synergy with IFN-gamma, whereas inhibition of the IFN-gamma-promoted IL-23 production. Collectively, the results suggest that iNKT cells modify the IL-12p70/IL-23 balance to enhance the IL-12p70-induced cell-mediated immunity and suppress the IL-23-dependent inflammatory pathologies. These results may account for the long-appreciated contrasting beneficial and adverse consequence of ligand activation of iNKT cells.

Role of human non-invariant NKT lymphocytes in the maintenance of type 2 T helper environment during pregnancy.

The molecular and cellular mechanisms that generate the T(h)2 cytokine environment necessary for the maintenance of pregnancy are still not fully understood. We herein show that the human decidua is highly enriched for TCR alpha beta(+)CD161(+) NKT cells. They express non-invariant antigen receptors encoded by diverse TCRV alpha- and V beta-chain gene segments, thereby referred to as non-invariant NKT (non-iNKT) cells. In spite of their diverse TCR expression, they do not recognize fetal allo-antigens but specifically responded to CD1d-transfected cell lines. In contrast to the peripheral blood non-iNKT cells, the decidua-residing non-iNKT cells had a marked T(h)2 bias. In addition, they suppress the mixed leukocyte reaction directed against the paternal antigens. The T(h)2 cytokines have been known to stimulate trophoblast outgrowth and invasion. Thereby, the non-iNKT cells residing in the decidual tissue may have a functionally important interaction with the villous and extravillous trophoblast cells expressing CD1d and may therefore play a pivotal role in successful pregnancy by inhibiting fetal rejection and enhancing placental growth. These findings may reflect one mechanism that is an essential component for the T(h)2 environment necessary for the maintenance of pregnancy.

Increased ovarian follicle atresia in obese Zucker rats is associated with enhanced expression of the forkhead transcription factor FOXO1.

It is well established that hyperinsulinemia, resulting from insulin resistance, plays a role in the pathophysiology of polycystic ovary syndrome (PCOS). The aim of this study was to investigate if ovarian follicular development and atresia are impaired in obese hyperinsulinemic (fa/fa) Zucker rats. To gain insight into the molecular mechanism of follicular atresia, we also examined the expression and localization of forkhead transcription factor FOXO1, a major regulator of cell fate decisions such as differentiation, cell-cycle arrest, and cell death. Serum insulin but not gonadotropin levels were significantly higher in obese (fa/fa) rats when compared to lean controls. Total ovarian follicle number and the percentage of atretic follicles were also significantly increased in obese (fa/fa) rats. Follicle atresia was associated with nuclear accumulation of FOXO1 transcription factor in TUNEL-positive granulosa cells. These results suggest a role for FOXO1 in granulosa cell apoptosis and increased ovarian follicle atresia associated with hyperinsulinemia.

17 Beta-estradiol (E2) plus tumor necrosis factor-alpha induces a distorted maturation of human monocyte-derived dendritic cells and promotes their capacity to initiate T-helper 2 responses.

There is growing evidence that 17 beta-estradiol (E2) modulates immune function. Recent studies indicated that certain effects of E2 on in vivo immune function are not a result of a direct action on T cells, but rather an indirect action on antigen-presenting cells. This study demonstrates that the pregnancy-associated doses of E2 plus tumor necrosis factor-alpha (TauNuF alpha) induce distorted maturation of human dendritic cells (DCs) that result in an increased capacity to induce T helper (Th) 2 responses. E2 did not affect the expression of human leukocyte antigen class II and costimulatory molecules by DCs, but elicited the ability of DC to produce CC chemokine ligand 1, which can attract CCR8-expressing Th2 cells and regulatory T cells. In addition, E2/TNF alpha-matured DCs increased the production of IL-10 relative to the IL-12p70 on CD40 ligation, thereby inducing naive T-cell differentiation into a Th2. Moreover, the increased concentration of E2 in the route of maturation did indeed further enhance Th2 deviation. The dominant Th2 deviation was induced at a high E2 concentration typical during pregnancy. These findings demonstrate that the high physiological levels of E2 may be an important endogenous component for regulating the DC function and skewing the immune response toward Th2.

Monitoring OH-PCBs in PCB transport worker's urine as a non-invasive exposure assessment tool.

In this study, we analyzed hydroxylated polychlorinated biphenyls (OH-PCBs) in urine of both PCB transport workers and PCB researchers. A method to monitor OH-PCB in urine was developed. Urine was solid-phase extracted with 0.1% ammonia/ methanol (v/v) and glucuronic acid/sulfate conjugates and then decomposed using ß-glucuronidase/arylsulfatase. After alkaline digestion/derivatization, the concentration of OH-PCBs was determined by HRGC/HRMS-SIM. In the first sampling campaign, the worker's OH-PCB levels increased several fold after the PCB waste transportation work, indicating exposure to PCBs. The concentration of OH-PCBs in PCB transport workers' urine (0.55~11 µg/g creatinine (Cre)) was higher than in PCB researchers' urine (< 0.20 µg/g Cre). However, also a slight increase of OH-PCBs was observed in the researchers doing the air sampling at PCB storage area. In the second sampling, after recommended PCB exposure reduction measures had been enacted, the worker's PCB levels did not increase during handling of PCB equipment. This suggests that applied safety measures improved the situation. Hydroxylated trichlorobiphenyls (OH-TrCBs) were identified as a major homolog of OH-PCBs in urine. Also, hydroxylated tetrachlorobiphenyls (OH-TeCBs) to hydroxylated hexachlorobiphenyls (OH-HxCBs) were detected. For the sum of ten selected major indicators, a strong correlation to total OH-PCBs were found and these can possibly be used as non-invasive biomarkers of PCB exposure in workers managing PCB capacitors and transformer oils. We suggest that monitoring of OH-PCBs in PCB management projects could be considered a non-invasive way to detect exposure. It could also be used as a tool to assess and improve PCB management. This is highly relevant considering the fact that in the next 10 years, approx. 14 million tons of PCB waste need to be managed. Also, the selected populations could be screened to assess whether exposure at work, school, or home has taken place.

Generation of mouse pluripotent stem cell-derived proliferating myeloid cells as an unlimited source of functional antigen-presenting cells.

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow-derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2K(b)-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs.

Discharge of perfluorinated compounds from rivers and their influence on the coastal seas of Hyogo prefecture, Japan.

The aim of this study was to investigate 12 perfluorinated compounds (PFCs) including perfluorinated carboxylates (C4-C12) and perfluorinated alkyl sulfonates (C4, C6, and C8) in river and seawater samples to determine contamination levels in the aquatic environment of Hyogo prefecture, Japan. High levels of perfluorohexanoic acid (PFHxA; 2300-16,000 ng/L) were detected in the Samondogawa River at Tatsumi Bridge downstream of a PFC production facility; this location also had the highest mass flow rate of PFCs (3900-29,000 kg/y). Widespread contamination of coastal waters was confirmed with PFHxA as the dominant compound. Perfluorooctanoic acid was also prevalent in coastal waters. The concentration of PFHxA in coastal seawater and the distance from the mouth of the Samondogawa River were inversely related. This discharge of high concentrations of PFHxA from the Samondogawa River may have affected concentrations of PFCs in Osaka Bay.

Polychlorinated biphenyl (118) activates osteoclasts and induces bone resorption in goldfish.

To analyze the effect of polychlorinated biphenyl (PCB) 118 on fish bone metabolism, we examined osteoclastic and osteoblastic activities, as well as plasma calcium levels, in the scales of PCB (118)-injected goldfish. In addition, effect of PCB (118) on osteoclasts and osteoblasts was investigated in vitro. Immature goldfish, in which the endogenous effects of sex steroids are negligible, were used. PCB (118) was solubilized in dimethyl sulfoxide at a concentration of 10 ppm. At 1 and 2 days after PCB (118) injection (100 ng/g body weight), both osteoclastic and osteoblastic activities, and plasma calcium levels were measured. In an in vitro study, then, both osteoclastic and osteoblastic activities as well as each marker mRNA expression were examined. At 2 days, scale osteoclastic activity in PCB (118)-injected goldfish increased significantly, while osteoblastic activity did not change significantly. Corresponding to osteoclastic activity, plasma calcium levels increased significantly at 2 days after PCB (118) administration. Osteoclastic activation also occurred in the marker enzyme activities and mRNA expressions in vitro. Thus, we conclude that PCB (118) disrupts bone metabolism in goldfish both in vivo and in vitro experiments.

Investigation of environmental contamination of mono-isopropylnaphthalene, di-isopropylnaphthalene and tri-isopropylnaphthalene in Hyogo in Japan.

Di-isopropylnaphthalene (DIPN) has highly persistent and bioaccumulative properties, and a large amount of DIPN is used as a PCB substitute in Japan. However, DIPN in the environment has not been thoroughly investigated. In addition, mono-isopropylnaphthalene (MIPN) and tri-isopropylnaphthalene (TIPN), which are the homologues of DIPN, have similar properties to DIPN. In this study, simultaneous analytical methods for MIPN, DIPN, and TIPN for air, environmental water, sediment, and biological samples were developed, and the resultant contamination caused by each in the environment was investigated. DIPN was detected at 1.1 ± 0.38 ng/m(3) in air and between < 1.9 and 9.8 ng/L in river water, but MIPN and TIPN were not. In Lateolabrax japonicas (Japanese sea perch), TIPN was detected from only females at between 0.65 and 1.4 ng/g-wet. DIPN was detected from all perches at between 1.2 and 3.4 ng/g-wet. DIPN and TIPN isomer fingerprints in females were different from those in the reference standard stock solution ones. In sediments, MIPN, DIPN, and TIPN were detected at between < 0.16 and 8.6 ng/g-dry, between < 1.1 and 4400 ng/g-dry, and between < 0.83 and 500 ng/g-dry, respectively. The contamination trend of DIPN in the sediments was similar to that of PCBs.

Human chorionic gonadotropin confers resistance to oxidative stress-induced apoptosis in decidualizing human endometrial stromal cells.

OBJECTIVE: To investigate the effect of hCG on the expression of genes involved in oxidative stress resistance observed in decidualizing human endometrial stromal cells (HESCs). DESIGN: In vitro experiment. SETTING: Saitama Medical University hospital. PATIENT(S): Premenopausal women undergoing hysterectomy for myoma uteri. INTERVENTION(S): HESCs from hysterectomy specimens were isolated and incubated with 8-bromo-cyclic adenosine monophosphate and medroxyprogesterone acetate in the presence or absence of recombinant hCG (rhCG) at various concentrations. Hydrogen peroxide was used as the source of reactive oxygen species (ROS). MAIN OUTCOME MEASURE(S): Apoptotic cells, FOXO1, superoxide dismutase 2 (SOD2), Bax, and Bcl-2 protein expression. RESULT(S): In a dose-dependent manner, rhCG conferred additional protection to decidualizing HESCs against oxidative stress-induced apoptosis. In parallel, rhCG augmented the expression of the forkhead transcription factor FOXO1 and its downstream target, the ROS scavenger SOD2. rhCG also inhibited the expression of the proapoptotic Bax protein and up-regulated antiapoptotic Bcl-2 levels in decidualizing HESCs exposed to ROS. CONCLUSION(S): The data suggest that hCG might improve the uterine environment upon implantation by suppressing apoptotic responses in the maternal decidua under oxidative stress.

Structural basis of species differences between human and experimental animal CYP1A1s in metabolism of 3,3',4,4',5-pentachlorobiphenyl.

Coplanar polychlorinated biphenyls included in dioxin-like compounds are bio-accumulated and adversely affect wildlife and human health. Although many researchers have studied the metabolism of PCBs, there have been few reports of the in vitro metabolism of 3,3',4,4',5-pentachlorobiphenyl (PCB126), despite the fact that it has the highest toxicity among PCB congeners. Cytochrome P450 (CYP) 1A1 proteins can metabolize some dioxins and PCBs by hydroxylation, but the activities of human and rat CYP1A1 proteins are very different. The mechanism remains unclear. From our results, rat CYP1A1 metabolized PCB126 into 4-OH-3,3',4',5-tetrachlorobiphenyl and 4-OH-3,3',4',5,5'-pentachlorobiphenyl, but human CYP1A1 did not metabolize. Homology models of the two CYP proteins, and docking studies, showed that differences in the amino acid residues forming their substrate-binding cavities led to differences in the size and shape of the cavities; only the cavity of rat CYP1A1 allowed PCB126 close enough to the haem to be metabolized. Comparison of the amino acid residues of other mammalian CYP1A1 proteins suggested that rats have a unique metabolism of xenobiotics. Our results suggest that it is necessary to be careful in human extrapolation of toxicity data estimated by using the rat as an experimental animal, especially in the case of compounds metabolized by CYP1A1.

Bisphenol A in combination with TNF-alpha selectively induces Th2 cell-promoting dendritic cells in vitro with an estrogen-like activity.

Bisphenol A (BPA) is a monomer used in manufacturing a wide range of chemical products, including epoxy resins and polycarbonate. BPA, an important endocrine disrupting chemical that exerts estrogen-like activities, is detectable at nanomolar levels in human serum worldwide. The pregnancy associated doses of 17beta-estradiol (E2) plus tumor-necrosis factor-alpha (TNF-alpha) induce distorted maturation of human dendritic cells (DCs) that result in an increased capacity to induce T helper (Th) 2 responses. The current study demonstrated that the presence of BPA during DC maturation influences the function of human DCs, thereby polarizing the subsequent Th response. In the presence of TNF-alpha, BPA treatment enhanced the expression of CC chemokine ligand 1 (CCL1) in DCs. In addition, DCs exposed to BPA/TNF-alpha produced higher levels of IL-10 relative to those of IL-12p70 on CD40 ligation, and preferentially induced Th2 deviation. BPA exerts the same effect with E2 at the same dose (0.01-0.1 microM) with regard to DC-mediated Th2 polarization. These findings imply that DCs exposed to BPA will provide one of the initial signals driving the development and perpetuation of Th2-dominated immune response in allergic reactions.

Distinct subsets of human invariant NKT cells differentially regulate T helper responses via dendritic cells.

Invariant natural killer T (iNKT) cells regulate the T helper (Th) 1/2 balance and elicit either enhancement or suppression of the immune responses. However, the exact mechanism by which iNKT cells exert these contrasting functions has remained elusive. We demonstrate herein that two major distinct subsets of human iNKT cells, CD4+CD8beta(-) (CD4+) and CD4(-)CD8beta(-) (double negative; DN) cells, express functional CD40 ligand (CD40L), but they differentially regulate the dendritic cell (DC) function by reciprocal NKT-DC interactions, thereby influencing the subsequent Th response. The CD4 subset stimulated by alpha-galactosylceramide (alpha-GalCer)-loaded DC immediately produced massive amounts of IL-4 and IL-13, which together with IFN-gamma enhanced CD40L-induced IL-12 production by DC. In contrast, the DN subset eliminated the DC by cytolysis and changed the living DC into a default subtype, in turn markedly down-regulating the levels of IL-12. Therefore, the DC stimulated by the CD4 subset preferentially induced Th1 responses, whereas the DC reacted with the DN subset induced a shift toward Th2 responses. These findings may provide an important insight into better understanding the contribution of iNKT-DC cross-talk governing the Th1/2 balance and the diverse influences of iNKT cells in various diseases.

Identification of functional type 1 ryanodine receptors in human dendritic cells.

Ryanodine receptor (RyR) is a Ca(2+) channel that mediates Ca(2+) release from intracellular stores. Altered Ca(2+) homeostasis in skeletal muscle which usually occurs as a result of point mutations in type 1 RyR1 (RyR1) is a key molecular event triggering malignant hyperthermia (MH). There are three RyR isoforms, and we herein show, for the first time, that human dendritic cells (DCs) preferentially express RyR1 mRNA among them. The RyR activator, 4-chloro-m-cresol (4CmC), induced Ca(2+) release in DCs, and this response was eliminated by dantrolene, an inhibitor of the RyR1, and was unaffected by xestospongin C, a selective inhibitor of IP(3) receptor. Activation of RyR1 reduced LPS-induced IL-10 production, promoted the expression of HLA-DR and CD86, and thereby exhibited an improved capacity to stimulate allogeneic T cells. These findings demonstrate that RyR1-mediated calcium signaling modifies diverse DC responses and suggest the feasibility of using DC preparations for the diagnosis of MH.