Effects of Components of PM2.5 Collected in Japan on the Respiratory and Immune Systems.

Epidemiologic studies have reported that particulate matter with aerodynamic diameters ≤2.5 µm (PM2.5) affect respiratory diseases, including asthma. The components and/or factors of PM2.5 that contribute to the exacerbation of asthma have not been identified. We investigated the effects of extracts of PM2.5 collected in Japan on the respiratory and immune systems. PM2.5 was collected from an industrial area and an urban area in December 2013. Airway epithelial cells and immune cells were exposed to aqueous or organic extracts of PM2.5. Exposure to extracts from both areas, especially to organic extracts rather than aqueous extracts, caused a pro-inflammatory response via interleukin (IL) 6 production from airway epithelial cells, and it induced the maturation/activation of bone marrow-derived antigen-presenting cells via dendritic and epithelial cell (DEC) 205 and cluster of differentiation (CD) 86 expression and proportional changes in the constitution of the splenocytes. The extracts collected from the industrial area tended to show greater effects than those from the urban area. These results suggest that organic components of PM2.5 affect the respiratory and immune systems. These effects can differ by the collection areas. In addition, IL-6, DEC205, and CD86 can be predictive biomarkers for the respiratory and immune effects of ambient PM2.5.

Modulation at a cellular level of the thyroid hormone receptor-mediated gene expression by 1,2,5,6,9,10-hexabromocyclododecane (HBCD), 4,4'-diiodobiphenyl (DIB), and nitrofen (NIP).

Previously, we demonstrated that some endocrine disrupting chemicals affected thyroid hormone receptor (TR)-mediated gene expression in HeLaTR cells that stably expressed the human TRalpha1. To examine whether widely used brominated flame retardants and pesticides affect TR-mediated gene expression, those with organohalogen, which is also present in T3, were screened. To monitor the TR-mediated gene expression, HeLaTR cells were transfected with a luciferase gene that was linked to the thyroid hormone responsive element. Thus, transcription of the luciferase gene in HeLaTR cells is driven by TR. By screening 38 chemical agents, it was found that 4,4'-diiodobiphenyl (DIB), markedly, and 1,2,5,6,9,10-hexabromocyclododecane (HBCD) and nitrofen (NIP), to a much lesser extent but significantly, enhanced the expression of the luciferase gene at concentrations that did not affect the growth of HeLaTR cells. DIB also augmented the E2-induced expression of the luciferase gene that was linked to the estrogen responsive element in MCF7 cells, whereas HBCD and NIP did not. These results indicate that DIB augments TR- and ER-mediated gene expression, but HBCD and NIP affect only TR-mediated gene expression. Thus, there is a potential risk that HBCD, DIB, and NIP act as endocrine disrupters in animals and human beings.

2,3,7,8-tetrachlorodibenzo-p-dioxin augments the modulation of gene expression mediated by the thyroid hormone receptor.

Previously, we reported on genes whose expression was highly modulated by T3 in the HeLaTR cells that stably expressed the thyroid hormone receptor (TR). In this study, we examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on TR-mediated gene expression. In the HeLaTR cells, T3 induced the expression of the reporter gene in a thyroid hormone responsible element (TRE)-dependent manner. When the cells were cultured in the presence of T3, the addition of TCDD but not 4-hydroxy-2',3,4',5,6'-pentachlorobiphenyl (PCB-OH), bisphenol A (BPA), or di(2-ethylhexyl)phthalate (DEHP) to the culture media further enhanced the T3-induced expression of the reporter gene. RT-PCR revealed that mRNA levels of 4-1BB, fmfc, PSCA, PSG7, RANTES, and TRAF1, which were highly increased by T3, were further elevated in cells exposed to T3 and TCDD. Also, the mRNA level of BMP6, which was decreased by T3, further declined in the cells exposed to both T3 and TCDD. In contrast to the effect of TCDD, PCB-OH suppressed the modulation of these gene expressions by T3. Neither TCDD nor PCB-OH alone affected the expression of 4-1BB, fmfc, PSCA, PSG7, RANTES, TRAF1, or BMP6. These results indicate that TCDD augments the cellular responses to T3 by hyperactivating TR-mediated gene expression, whereas PCB-OH suppresses cellular responses to T3 by negatively regulating it. Based on these findings, enzyme-linked immunosorbent assay (ELISA) for the PSCA protein in the HeLaTR cells was established. Such assays will be useful to monitor the effects of endocrine disrupting chemicals (EDCs) on TR-mediated gene expression.

Dioxin-like potencies and extractable organohalogens (EOX) in medical, municipal and domestic waste incinerator ashes in Japan.

Ash samples collected from medical, municipal and small-scale domestic incinerators in Japan were tested for dioxin-like activity using bioassay technique (ethoxyresorufin-O-deethylase: EROD assay) and for extractable organohalogens (EOX) using instrumental neutron activation analysis in order to estimate potential toxicity and responsible chemicals in those samples. Crude extracts and fractions cleaned-up for dioxin analysis from the samples were used for the analysis. The ranges of dioxins in the ashes were between 2.23 and 12.29 ng TEQ/g (dry weight). Relative potency ranges estimated by EROD assay in the medical incinerator ashes were 3.8-17.6 times higher than the results of conventional chemical analysis. EOX analysis suggested that ash samples contained plenty of organochlorine compounds apart from chlorinated dioxins. In addition, medical waste incinerator ashes were considered to have relatively higher amount of organoiodine compounds. In the cleaned-up fractions, bioassay potency ranges were lower than those in the crude extracts. However, some samples still exhibited higher potency than expected from chemical analysis. Though some polycyclic aromatic hydrocarbons were found in the fractions, the amounts were relatively low (0.39-10.56 ng/g). The results imply that some bioactive organohalogens that cannot be detected in the conventional chemical analysis might have potential for dioxin-like toxicity, and contribute to higher bioassay activities. The combination of the chemical analysis with the bioassay and EOX provides rough figure of dioxin-like toxicity and suggests types of organohalogen compounds that should be identified as a part of dioxin analysis for control emission from an incineration plant.

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system.

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.

The Candida albicans gene for mRNA 5-cap methyltransferase: identification of additional residues essential for catalysis.

The 5'-cap structure of eukaryotic mRNA is methylated at the terminal guanosine by RNA (guanine-N7-)-methyltransferase (cap MTase). Saccharomyces cerevisiae ABD1 (ScABD1) and human hMet (also called CMT1) genes are responsible for this enzyme. The ABD1 homologue was cloned from the pathogenic fungus Candida albicans and named C. albicans ABD1 (CaABD1). When expressed as a fusion with glutathione S-transferase (GST), CaAbd1p displayed cap MTase activity in vitro and rescued S. cerevisiae abd1delta null mutants, indicating that CaABD1 specifies an active cap MTase. Although the human cap MTase binds to the human capping enzyme (Hce1p), which possesses both mRNA guanylyltransferase (mRNA GTase) and mRNA 5'-triphosphatase (mRNA TPase) activities, yeast two-hybrid analysis demonstrated that in yeast neither mRNA GTase nor mRNA TPase physically interacted with the Abd1 protein. Comparison of the amino acid sequences of known and putative cap MTases revealed a highly conserved amino acid sequence motif, Phe/Val-Leu-Asp/Glu-Leu/Met-Xaa-Cys-Gly-Lys-Gly-Gly-Asp-Leu-Xaa-Lys, which encompasses the sequence motif characteristic of divergent methyltransferases. Mutations in CaAbd1p of leucine at the second and the twelfth positions (so far uncharacterized) to alanine severely diminished the enzyme activity and the functionality in vivo, whereas those of leucine at the fourth, cysteine at the sixth, lysine at the eighth, and glycine at the tenth positions did not. Furthermore, valine substitution for the twelfth, but not for the second, leucine in that motif abolished the activity and functionality of CaAbd1p. Thus, it appears that leucine at the second and the twelfth positions in the motif, together with a previously identified acidic residue in the third, glycine at the sixth and glutamic acid at the eleventh positions, play important roles in the catalysis, and that side chain length is crucial for the activity at the twelfth position in the motif.