Yoshihisa Tomioka

Neurobehavioral effects of tetrabromobisphenol A, a brominated flame retardant, in mice

Tetrabromobisphenol A (TBBPA) is widely used as a flame retardant and is suspected to be stable in the environment with possible widespread human exposures. In the present study, we investigated the behavioral effects of TBBPA and measured the levels of TBBPA in the brain after oral administration in mice. Acute treatment with TBBPA (5mg/kg body weight) 3h before the open-field test induced an increase in the horizontal movement activities. In contextual fear conditioning paradigm, mice treated with TBBPA (0.1mg/kg or 5mg/kg body weight) showed more freezing behavior than vehicle-treated mice. In addition, TBBPA (0.1mg/kg body weight) significantly increased the spontaneous alternation behavior in the Y-maze test. The levels of TBBPA in the brain following TBBPA treatment were determined by using LC/ESI-MS/MS system. In the brain regions examined, high amounts of TBBPA were detected in the striatum after treatment with 0.1mg/kg or 5mg/kg body weight TBBPA, whereas non-specific accumulation of TBBPA in the brain was found after treatment with 250 mg/kg body weight TBBPA. These results suggest that TBBPA accumulates in brain regions including the striatum and induces the behavioral alterations. Together, the possibility of widespread human exposure to TBBPA warrants further studies to characterize its neurotoxicity.

Mechanisms of mucin production by rhinovirus infection in cultured human airway epithelial cells.

Mucus hypersecretion relates to exacerbations of bronchial asthma and chronic obstructive pulmonary disease (COPD) caused by rhinovirus (RV) infection. We examined the mechanisms of RV infection-induced mucin production in human tracheal surface epithelial cells and submucosal gland cells. RV14 up-regulated the mRNA expression of MUC2, MUC3, MUC5AC, MUC5B and MUC6, and increased MUC5AC and total mucin concentration in supernatants and lysates of the surface cells. An inhibitor of the nuclear factor kappaB caffeic acid phenylethyl ester, inhibitors of selective p44/42 mitogen-activated protein kinase-kinase PD98059 and U0126, and a selective Src inhibitor PP1 attenuated MUC5AC mRNA expression, and secretion and production of MUC5AC and total mucin glycoprotein in the surface cells. In the gland cells, RV14 also increased mRNA expression of MUC2, MUC5AC, MUC5B and MUC7, and the inhibitors attenuated the secretion of total mucin glycoprotein. Src-related p44/42 mitogen-activated protein kinase pathway may be associated with RV-induced mucin hypersecretion in human airways.

Swine Toll-like receptor 9 recognizes CpG motifs of human cell stimulant

Complementary DNA (cDNA) encoding swine Toll-like receptor 9 (sTLR9) was isolated from Peyers patches (Pps) of gut-associated lymphoid tissue (GALT). The complete open reading frame (ORF) of sTLR9 contains 3093 bp coding deduced 1030 amino acid residues. The amino acid sequence of sTLR9 was characterized by a signal peptide followed by multiple leucine-rich repeats, a transmembrane sequence and a cytoplasmic domain homologous to that of the human interleukin-1 receptor (TIR). The sTLR9 showed a higher amino acid identity with humans (81.8%) and felis catus (86.7%) than mice (74.9%). The HEK293T cells transfected with pCXN2.1-FLAG DNA containing the sTLR9 cDNA were expressed sTLR9 as a membrane-bound molecules, which were reactive with anti-sTLR9 rabbit polyclonal antibody. Moreover, the transfectant was responsible for the CpG oligo DNA. sTLR9 was preferentially expressed in Pps and mesenteric lymph nodes (MLNs), and its degree was approximately three times higher than a spleen but weak in the other tissues by the real-time quantitative PCR analyses. The strong expression of sTLR9 in Pps and MLNs and its recognizing CpG DNA for human cell stimulant are shown first in this study, which may help in understanding the intestinal immune system mediated by a bacterial DNA through TLR9.

Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD

The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis-mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment.

Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS.

Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids (GPs). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs.

Conformational Change in Transfer RNA Is an Early Indicator of Acute Cellular Damage

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

Simultaneous quantification of prostaglandins, isoprostane and thromboxane in cell-cultured medium using gas chromatography–mass spectrometry

We have developed a simultaneous quantification method for prostaglandin (PG) E(2), PGD(2), PGF(2 alpha), 8-epi-PGF(2 alpha), 6-keto-PGF(1 alpha) and thromboxane (TX) B(2). Using [3,3,4,4-(2)H(4)]PGE(2), [3,3,4,4-(2)H(4)]PGD(2), [3,3,4,4-(2)H(4)]8-epi-PGF(2 alpha), [3,3,4,4-(2)H(4)]PGF(2 alpha), [3,3,4,4-(2)H(4)]6-keto-PGF(1 alpha) and [18,18,19,19-(2)H(4)]TXB(2) as internal standards (I.S.), the eicosanoids and their I.S. were simultaneously extracted by solid-phase extraction from cell-cultured medium, derivatized to methyl ester/methoxim/tert.-butyldimethylsilyl ether derivatives and analyzed using gas chromatography-mass spectrometry in the selected ion monitoring mode. The accuracy for the added eicosanoids ranged from 92 to 113%, and coefficients of variation ranged from 0.1 to 12.2%. Increased eicosanoids in RAW264.7 and U937 cells stimulated by lipopolysaccharide were suppressed by NS-398 and indometacin. This simultaneous quantification method can be applied routinely for assaying eicosanoids in vitro.

Concise synthesis of ciguatoxin ABC-ring fragments and surface plasmon resonance study of the interaction of their BSA conjugates with monoclonal antibodies.

Monoclonal antibodies (mAbs), 4H2 and 6H7, were prepared previously using a protein conjugate of a 1:1 epimeric mixture of the synthetic ABC-ring fragments of ciguatoxin (CTX), 3 and 4. Here, the interactions of these mAbs with the fragments of CTX and CTX3C, 3 and 5, were investigated by surface plasmon resonance (SPR) spectroscopy in an attempt to clarify an antigenic determinant. Compared with the previous synthesis, the fragment 3 possessing the 2S configuration was synthesized from tri-O-acetyl-D-glucal much more effectively. The mAb 4H2 was already known to show a dose-dependent binding to the bovine serum albumin (BSA) conjugate of 3, but not to that of 5. The present SPR study of 4H2 demonstrates that the A-ring side chain of 3 plays a decisive role as an epitope. Therefore, SPR can effectively replace the ELISA method for the analysis of mAbs.

A distinct regulatory role of Th17 cytokines IL-17A and IL-17F in chemokine secretion from lung microvascular endothelial cells.

Th17 cytokines IL-17A and IL-17F play a critical role in the activation and recruitment of neutrophils at airway inflammation mainly through the induction of CXC chemokines in the lungs. Vascular endothelial cells belong to the category of major CXC chemokine-producing cells. However, until now, the precise role of Th17 cytokines in CXC chemokine secretion in lung microvascular endothelial cells (LMVECs) has not been fully elucidated. In this study, we examined the biological effects of Th17 cytokines IL-17A and IL-17F on CXCL1, CXCL5, and CXCL8 release in LMVECs. Both IL-17 receptor A (IL-17RA) and IL-17RC are expressed on the surface of LMVECs. In contrast to IL-17F, IL-17A significantly upregulated CXCL1 mRNA expression and protein release, whereas both IL-17A and IL-17F did not have the ability to induce CXCL5 and CXCL8 secretion in LMVECs. IL-17A and IL-17F displayed positive regulatory effects on IL-1β-induced CXCL1, CXCL5, and CXCL8 secretion. On the other hand, IL-17A enhanced the upregulating effect of TNF-α on CXCL1, CXCL5, and CXCL8 release, whereas IL-17F had a negative regulatory effect on TNF-α-mediated secretion. Th2 cytokines IL-4 and IL-13 showed an inhibitory effect on IL-1β plus IL-17A-induced CXCL1, CXCL5, and CXCL8 secretion, but displayed a positive regulatory effect on TNF-α plus IL-17A-induced secretion. These results provide evidence that Th17 cytokines IL-17A and IL-17F have a distinct regulatory role in CXCL1, CXCL5, and CXCL8 expression in LMVECs stimulated either with IL-1β or with TNF-α. Our findings also suggest that CXC chemokine secretion in LMVECs may be complicatedly regulated by Th17 cytokines, Th2 cytokines, and macrophage-associated cytokines in pathological conditions such as bronchial asthma.

Functional Characterization of CYP2B6 Allelic Variants in Demethylation of Antimalarial Artemether

Artemether (AM) is one of the most effective antimalarial drugs. The elimination half-life of AM is very short, and it shows large interindividual variability in pharmacokinetic parameters. The aim of this study was to identify cytochrome P450 (P450) isozymes responsible for the demethylation of AM and to evaluate functional differences between 26 CYP2B6 allelic variants in vitro. Of 14 recombinant P450s examined in this study, CYP2B6 and CYP3A4 were primarily responsible for production of the desmethyl metabolite dihydroartemisinin. The intrinsic clearance (Vmax/Km) of CYP2B6 was 6-fold higher than that of CYP3A4. AM demethylation activity was correlated with CYP2B6 protein levels (P = 0.004); however, it was not correlated with CYP3A4 protein levels (P = 0.27) in human liver microsomes. Wild-type CYP2B6.1 and 25 CYP2B6 allelic variants (CYP2B6.2-CYP2B6.21 and CYP2B6.23-CYP2B6.27) were heterologously expressed in COS-7 cells. In vitro analysis revealed no enzymatic activity in 5 variants (CYP2B6.8, CYP2B6.12, CYP2B6.18, CYP2B6.21, and CYP2B6.24), lower activity in 7 variants (CYP2B6.10, CYP2B6.11, CYP2B6.14, CYP2B6.15, CYP2B6.16, CYP2B6.20, and CYP2B6.27), and higher activity in 4 variants (CYP2B6.2, CYP2B6.4, CYP2B6.6, and CYP2B6.19), compared with that of wild-type CYP2B6.1. In kinetic analysis, 3 variants (CYP2B6.2, CYP2B6.4, and CYP2B6.6) exhibited significantly higher Vmax, and 3 variants (CYP2B6.14, CYP2B6.20 and CYP2B6.27) exhibited significantly lower Vmax compared with that of CYP2B6.1. This functional analysis of CYP2B6 variants could provide useful information for individualization of antimalarial drug therapy.