Liver injury induced by thirty- and fifty-nanometer-diameter silica nanoparticles.

Nano-size silica material is a promising reagent for disease diagnosis, cosmetics, and the food industry. For the successful application of nanoparticle materials in bioscience, evaluation of nano-size material toxicity is important. We previously found that nano-size silica particles caused acute liver failure in mice. However, the hepatotoxicity of nanosilica particles with the diameter of 70 nm or less is unknown. Here, we investigated the relationship between particle size and toxicity using nanosilica particles with diameters of 30, 50, and 70 nm (SP30, SP50, and SP70, respectively). We observed dose-dependent increases in hepatic injury following administration of SP50 and SP30, with SP30 causing greater acute liver injury than that seen with SP50. Smaller silica nanoparticles induced liver injury even at proportionally lower dose levels. Furthermore, we investigated the combinatorial toxicity of SP30 in the presence of chemically induced liver injury (including that caused by carbon tetrachloride, paraquat, cisplatin, and acetaminophen). We observed that particles of the smallest size tested (SP30) synergized with chemical substances in causing liver injury. These data suggest that the size (diameter) of the silica nanoparticles affects the severity of nanoparticle-induced liver injury, a finding that will be useful for future investigations in nanotechnology and nanotoxicology.

Deficient of a clock gene, brain and muscle Arnt-like protein-1 (BMAL1), induces dyslipidemia and ectopic fat formation.

A link between circadian rhythm and metabolism has long been discussed. Circadian rhythm is controlled by positive and negative transcriptional and translational feedback loops composed of several clock genes. Among clock genes, the brain and muscle Arnt-like protein-1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) play important roles in the regulation of the positive rhythmic transcription. In addition to control of circadian rhythm, we have previously shown that BMAL1 regulates adipogenesis. In metabolic syndrome patients, the function of BMAL1 is dysregulated in visceral adipose tissue. In addition, analysis of SNPs has revealed that BMAL1 is associated with susceptibility to hypertension and type II diabetes. Furthermore, the significant roles of BMAL1 in pancreatic ß cells proliferation and maturation were recently reported. These results suggest that BMAL1 regulates energy homeostasis. Therefore, in this study, we examined whether loss of BMAL1 function is capable of inducing metabolic syndrome. Deficient of the Bmal1 gene in mice resulted in elevation of the respiratory quotient value, indicating that BMAL1 is involved in the utilization of fat as an energy source. Indeed, lack of Bmal1 reduced the capacity of fat storage in adipose tissue, resulting in an increase in the levels of circulating fatty acids, including triglycerides, free fatty acids, and cholesterol. Elevation of the circulating fatty acids level induced the formation of ectopic fat in the liver and skeletal muscle in Bmal1 -/- mice. Interestingly, ectopic fat formation was not observed in tissue-specific (liver or skeletal muscle) Bmal1 -/- mice even under high fat diet feeding condition. Therefore, we were led to conclude that BMAL1 is a crucial factor in the regulation of energy homeostasis, and disorders of the functions of BMAL1 lead to the development of metabolic syndrome.

Aryl hydrocarbon receptor functions as a potent coactivator of E2F1-dependent trascription activity.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Several reports have shown that the AhR plays an important role in the control of cell-cycle progression, and this function is thought to be partly associated with the tumor promotion activity of dioxin. However, the underling mechanisms are not fully understood. We have previously shown that overexpression of AhR, as well as AhR ligand treatment, stimulates cell proliferation of human lung cancer A549 cells. In AhR-activated cells, the expression levels of DNA synthesis-related genes such as proliferating cell nuclear antigen (PCNA) and RFC38 are notably increased. Expression of these genes is mainly regulated by E2F1, a transcription factor that is crucial for transition of the cell cycle from G1 to S phase. We show here that the transcriptional activity of E2F1 is increased by the AhR agonist treatment and that this effect depends on the presence of AhR. Functional mapping of AhR showed that the Per-Arnt-Sim (PAS) B domain is required for promotion of E2F1 activity. The mechanism involves formation of a complex of AhR and E2F1 on the regulatory region in the E2F1 target gene, followed by recruitment of coactivator activator for thyroid hormone and retinoid receptors (ACTR). Consequently, the results in this study indicate the physiological function of AhR as a potent transcriptional coactivator of E2F1-dependent transcription and implicate the AhR-E2F1 interaction as a part of the mechanism by which AhR/Arnt promotes cell proliferation.

Comparative study of hydrogen peroxide- and 4-hydroxy-2-nonenal-induced cell death in HT22 cells.

Several studies have indicated that lipid peroxidation often occurs in response to oxidative stress, and that many aldehydic products including 4-hydroxy-2-nonenal (HNE) are formed when lipid hydroperoxides break down. In order to clarify the mechanism of oxidative stress-induced neuronal death in the nervous system, we investigated H(2)O(2)- and HNE-induced cell death pathways in HT22 cells, a mouse hippocampal cell line, under the same experimental conditions. Treatment with H(2)O(2) and HNE decreased the viability of these cells in a time- and concentration-dependent manner. In the cells treated with H(2)O(2), significant increases in the immunoreactivities of DJ-1 and nuclear factor-kappaB (NF-kappaB) subunits (p65 and p50) were observed in the nuclear fraction. H(2)O(2) also induced an increase in the intracellular concentration of Ca(2+), and cobalt chloride (CoCl(2)), a Ca(2+) channel inhibitor, suppressed the H(2)O(2)-induced cell death. In HNE-treated cells, none of these phenomena were observed; however, HNE adduct proteins were formed after exposure to HNE, but not to H(2)O(2). N-Acetyl-L-cysteine (NAC) suppressed both HNE-induced cell death and HNE-induced expression of HNE adduct proteins, whereas H(2)O(2)-induced cell death was not affected. These findings suggest that the mechanisms of cell death induced by H(2)O(2) different from those induced by HNE in HT22 cells, and that HNE adduct proteins play an important role in HNE-induced cell death. It is also suggested that the pathway for H(2)O(2)-induced cell death in HT22 cells does not involve HNE production.

Characterization of the molecular clock in mouse peritoneal macrophages.

Macrophages play essential roles in the innate immune system. In this study, we show that macrophage functions such as phagocytosis and cytokine/chemokine expressions display a circadian rhythm that is regulated by a molecular clock. Phagocytosis, a crucial early reaction by which macrophages protect their host against foreign particles, exhibited a circadian variation that peaks during the light period and bottoms during the dark period. These diurnal changes of phagocytosis activity in macrophages were induced without exogenous stimulants such as bacterial infection. The expression of the clock genes including brain and muscle Arnt-like protein-1 (BMAL1) exhibited robust circadian rhythms in macrophages. The expression patterns of the clock genes in macrophages were similar to those in the suprachiasmatic nucleus and other peripheral tissues. Among inflammation factors examined, the level of monocyte chemoattractant protein-1 (MCP-1/JE) mRNA exhibited most robust circadian oscillation. Expression of other cytokines such as IL-1beta, IL-6 and TNFalpha showed mild diurnal changes. Knockdown of the BMAL1 expression resulted in a decrease of the MCP-1/JE mRNA level in macrophages. BMAL1 increased significantly but weakly MCP-1/JE promoter activity. MCP-1/JE promoter activity is known to be regulated by nuclear factor-kappa B (NF-kappaB). NF-kappaB activity in BMAL1 knockdown macrophages was lower than that in control cells. Consequently, the circadian expression of MCP-1/JE in macrophages is regulated by BMAL1 through the activation of NF-kappaB. The results obtained in this study indicate that the innate immunoreactions involving macrophages are at least partly regulated by the autonomous clock machinery.

Transcriptional regulation of the hypoxia inducible factor-2alpha (HIF-2alpha) gene during adipose differentiation in 3T3-L1 cells.

Adipose differentiation is regulated by coordination of several signaling pathways and transcription factors. We recently showed that Hypoxia inducible factor-2alpha (HIF-2alpha) plays several supporting roles in adipose differentiation and adipocytes functions including regulation of glucose uptake followed by lipid synthesis. HIF-2alpha expression is increased during adipogenesis, indicating that its up-regulation is necessary for execution of adipogenesis and maintenance of mature adipocytes functions. Therefore, in this study, to understand the mechanism by which HIF-2alpha expression is induced during adipogenesis, we investigated the promoter activity of HIF-2alpha gene during adipogenesis in 3T3-L1 cells. A comparison of HIF-2alpha promoter activity between preadipocytes and adipocytes revealed that the sequence -478/-445 is the putative core element that contributes to differentiation-dependent up-regulation of HIF-2alpha promoter activity. Electrophoretic mobility shift assays showed the presence of the specific nuclear factor bound to the sequence -478/-445 in both preadipocytes and adipocytes. Computer analysis revealed that this element contains several Sp1/Sp3 binding sites. Indeed, the presence of Sp1/Sp3 consensus oligonucleotides diminished the formation of the complexes composed of the sequence -478/-445 and the nuclear factor. Furthermore, specific retarded bands were supershifted with anti-Sp1 or -Sp3 antibodies. Binding of Sp1 and Sp3 to this element was also confirmed by chromatin immunoprecipitation analysis. The element encompassing -478/-445 favors Sp3 in preadipocytes and Sp1 in adipocytes. Finally, the activity of -478/-445 was increased by Sp1 but decreased by Sp3. Consequently, these results suggest that Sp1 and Sp3 are involved in transcriptional regulation of HIF-2alpha expression during adipogenesis in 3T3-L1 cells.

Evaluation of some halogen biocides using a microbial biofilm system.

A simple method for the formation of microbial biofilms of three species, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Klebsiella pneumoniae, on a small glass slide was established, and its suitability for evaluation of disinfectant efficacy was examined. The biofilms formed were observed in situ by confocal laser scanning microscopy (CLSM). Using the biofilms established, biocidal efficacy of several halogen biocides, such as hypochlorite (HOCl), bromochlorodimethylhydantoin (Br, Cl-DMH), ammonia monochloramine (NH2Cl), a stabilized hypobromite biocide named STABREX, and a mixed solution of NH4Br and HOCl, was evaluated. The formation of NHBrCl in the mixed solution was indicated by UV spectra analysis. Biofilm cells were more resistant to these biocides than planktonic cells and the extent of resistance varied with the biocide tested. Among the biocides tested, the biocidal potency of HOCl was the most susceptible to the change brought about by biofilm formation. By CLSM observation, differences in biofilm conformation were revealed between the microbial species. The efficacy of the biocide tested varied with the structure of biofilms formed. The assay method developed in the present study would be useful for further investigation on biofilm disinfection.

Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis.

Brain and muscle Arnt-like protein-1 (BMAL1; also known as MOP3 or Arnt3) is a transcription factor known to regulate circadian rhythm. Here, we established its involvement in the control of adipogenesis and lipid metabolism activity in mature adipocytes. During adipose differentiation in 3T3-L1 cells, the level of BMAL1 mRNA began to increase 4 days after induction and was highly expressed in differentiated cells. In white adipose tissues isolated from C57BL/6J mice, BMAL1 was predominantly expressed in a fraction containing adipocytes, as compared with the stromal-vascular fraction. BMAL1 knockout mice embryonic fibroblast cells failed to be differentiated into adipocytes. Importantly, adding BMAL1 back by adenovirus gene transfer restored the ability of BMAL1 knockout mice embryonic fibroblast cells to differentiate. Knock-down of BMAL1 expression in 3T3-L1 cells by an RNA interference technique allowed the cells to accumulate only minimum amounts of lipid droplets in the cells. Adenovirus-mediated expression of BMAL1 in 3T3-L1 adipocytes resulted in induction of several factors involved in lipogenesis. The promoter activity of these genes was stimulated in a BMAL1-dependent manner. Interestingly, expression of these factors showed clear circadian rhythm in mice adipose tissue. Furthermore, overexpression of BMAL1 in adipocytes increased lipid synthesis activity. These results indicate that BMAL1, a master regulator of circadian rhythm, also plays important roles in the regulation of adipose differentiation and lipogenesis in mature adipocytes.

Occurrence and production of chloramines in the chlorination of creatinine in aqueous solution.

Occurrence and production of stable chloramines in the chlorination of creatinine, a constituent of perspiration and urine, in aqueous media were studied. Creatinine (5 x 10(-5)M) was treated with free chlorine in aqueous solutions at molar ratios of 0.5-8 (chlorine/creatinine) at pH 7.0 at room temperature for several days. At lower ratios of chlorine, two stable N-chlorocreatinine derivatives, which were determined as dichloramine fractions by the DPD method, were isolated by HPLC and identified by EI-MS and (1)H-NMR. One was 2-chloroamino-1-methylimidazolin-4-one (creatinine chloramine) and the other was 2-chloroamino-5-hydroxy-1-methylimidazolin-4-one (hydroxycreatinine chloramine). In addition, the formation of methylamine was identified by GC-MS analyses of its imine derivative formed with pentafluorobenzaldehyde. Methylamine forms stable chloramines, which might be determined as mono- and/or di-chloramine fractions together with free chlorine by the DPD method in the reaction mixtures at higher molar ratios of chlorine. In practice, small amounts of methylamine (ca. 19 microg/L) were detected in water samples collected from several swimming pools. Hence, methylamine may be an origin of elusive organic chloramine formed in the chlorination of swimming pools. A probable mechanism of the occurrence and processing of chlorination products of creatinine is suggested.

EPAS1 promotes adipose differentiation in 3T3-L1 cells.

Adipose differentiation is regulated by several transcription factors, such as the CAAT/enhancer-binding protein family and peroxisome proliferator activator (PPAR) gamma2. Several recent studies have shown that the basic helix-loop-helix-PAS superfamily is also involved in the regulation of adipose differentiation. In this study, we investigated the roles played by EPAS1 (endothelial PAS domain protein 1) in adipogenesis. EPAS1, also referred to as hypoxia-inducible factor 2alpha, is a transcription factor known to play essential roles in catecholamine homeostasis, vascular remodeling, and the maintenance of reactive oxygen species, and so forth. During adipose differentiation in 3T3-L1 cells, the level of EPAS1 mRNA began to increase 6 days after the induction, and EPAS1 was highly expressed in differentiated cells. To examine whether EPAS1 is involved in adipogenesis, we first isolated stable clones from 3T3-L1 cells in which we could induce the expression of an EPAS1 C-terminal deletion mutant (designated EPAS1-(1-485)) with the insect hormone. The induction of EPAS1-(1-485) allowed the cells to accumulate only minimum amounts of intracellular lipid droplets. Consistent with the morphological observations, a minimum amount of aP2 and PPARgamma2 mRNA was induced in the EPAS1-(1-485) cells. We then examined whether or not EPAS1 was able to promote adipogenesis in NIH 3T3 cells, a relatively nonadipogenic cell line. Overexpression of EPAS1 in NIH 3T3 cells induced a significant amount of lipid accumulation compared with that of the control cells in the presence of the PPARgamma ligand. The results were also confirmed by measuring the expression of adipocyte-related genes. Adenovirus-mediated EPAS1-(1-485) expression resulted in the reduction of basal and insulin-dependent glucose transport in 3T3-L1 adipocytes. The mechanism involved the transcriptional regulation of GLUT1, GLUT4, and IRS3 expression by EPAS1. Taken together, these results suggest that EPAS1 plays several supporting roles in maintaining specific aspects of adipogenesis and adipocyte function including regulation of glucose uptake followed by lipid synthesis.

Development of a headspace GC/MS analysis for carbonyl compounds (aldehydes and ketones) in household products after derivatization with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine.

Carbonyl compounds (aldehydes and ketones) are suspected to be among the chemical compounds responsible for Sick Building Syndrome and Multiple Chemical Sensitivities. A headspace gas chromatography/mass spectrometry (GC/MS) analysis for these compounds was developed using derivatization of the compounds into volatile derivatives with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBOA). For GC/MS detection, two ionization modes including electron impact ionization (EI) and negative chemical ionization (NCI) were compared. The NCI mode seemed to be better because of its higher selectivity and sensitivity. This headspace GC/MS (NCI mode) was employed as analysis for aldehydes and ketones in materials (fiber products, adhesives, and printed materials). Formaldehyde was detected in the range of N.D. (not detected) to 39 microg/g; acetaldehyde, N.D. to 4.1 microg/g; propionaldehyde, N.D. to 1.0 microg/g; n-butyraldehyde, N.D. to 0.10 microg/g; and acetone, N.D. to 3.1 microg/g in the samples analyzed.

Transcriptional regulation of the AhR gene during adipose differentiation.

The aryl hydrocarbon receptor (AhR) mediates a spectrum of toxicological and biological effects of dioxins. Studies on tissue distribution of the AhR in developing and adult animals demonstrated that the AhR is expressed in a tissue-specific and developmentally specific manner. Also, the expression level of the AhR in culture cells varies more than 50-fold among cell lines. Although the mode of AhR action has been studied extensively, the events that control the expression of the AhR gene itself are still poorly understood. We previously showed that the AhR protein is depleted during adipose differentiation, resulting in the loss of functional response to xenobiotics. In this study, to understand the mechanism by which the AhR is depleted during adipogenesis, we analyzed the AhR promoter activity during adipose differentiation in 3T3-L1 cells. Nuclear run-on assay revealed that the downregulation of the AhR during adipogenesis is primarily at the transcriptional level. To identify the sequence of the AhR promoter region responsible for differentiation-dependent suppression of AhR transcription, a series of deletion constructs linked to the CAT reporter were transfected into 3T3-L1 cells. A comparison of CAT activity between preadipocytes and adipocytes revealed that the sequence -378/-359 is core contributor to differentiation-dependent downregulation of AhR promoter activity. EMSA and UV crosslinking studies showed the presence of the factor bound to the sequence -378/-359. The binding activity was apparently higher in preadipocytes than in adipocytes. Consequently, the downregulation of the trans-acting factor may result in the suppression of AhR gene transcription during adipose differentiation.

Overexpression of the aryl hydrocarbon receptor (AhR) accelerates the cell proliferation of A549 cells.

The arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the physiological ligand for the AhR has not yet been identified, several reports have suggested that the AhR may play important roles not only in the regulation of xenobiotic metabolism but also in the maintenance of homeostatic functions [Singh et al. (1996) Arch. Biochem. Biophys. 329, 47-55; Crawford et al. (1997) Mol. Pharmacol. 52, 921-927; Chang et al. (1998) Mol. Cell. Biol. 18, 525-535]. Several lines of evidence suggest that one of the possible physiological roles of the AhR is regulation of cell proliferation. In this study, we first showed that treatment of A549 cells with the AhR agonist stimulates cell proliferation. The effect was antagonized by co-treatment with alpha-naphthoflavone. To obtain direct evidence that the AhR regulates cell proliferation, we isolated the clones that overexpress the AhR. These clones grow faster than control cells, and the rate of growth is proportional to the amount of the AhR. Cell cycle analysis revealed that the acceleration of cell growth by overexpression of the AhR is most probably due to shortening of the late M to S phases. Studies on the expression profiles of cell cycle regulators showed that the AhR or AhR ligand induces the expression of DP2, PCNA, and RFC38. DP2 is the transcription factor that forms the functional dimer with E2F and regulates the expression of several genes involved in DNA synthesis. Interestingly, both PCNA and RFC38 are target genes of E2F and the DP complex. Also, both of these factors are involved in regulating DNA polymerase delta activity. E2F activity was substantially increased in both the AhR-overexpressing cells and the AhR-agonist treated cells, suggesting that AhR-activated E2F/DP2 may induce the expression of PCNA and RFC38 and subsequent DNA synthesis. Down-regulation of the expression of the Arnt by RNAi diminished the effects of the AhR on the cell proliferation of the A549 cells. Consequently, we conclude that the AhR, presumably in collaboration with the Arnt, activates the DNA synthesis and the subsequent cell proliferation in A549 cells.

Effects of isocyanuric acid on the monochlorodimedone chlorinating rates with free chlorine and ammonia chloramine in water.

Changes in monochlorodimedone (MCD) chlorinating rates with free chlorine (mixture of HOCl and OCl-) and ammonia monochloramine (NH2Cl) in water at pH 7 by the addition of isocyanuric acid (H3Cy) were determined at room temperature. Decreases in MCD absorbance at 290nm in equimolar (0.04mM) reactions of MCD and free available chlorine solutions containing H3Cy (0.01-1.60 mM) were recorded in a stopped-flow spectrophotometer. The rates indicate second-order reactions. Since the rate with free chlorine was high (> 7.6 x 10(6) M(-1) s(-1)), the amounts of free chlorine in the solutions could be distinguished from that of chlorinated cyanurates. The chlorinating rates with chlorinated cyanurates decreased with an increase in H3Cy concentrations. Plotting the rates against the molar ratio of chlorine to H3Cy showed a linear correlation and the rates with chlorinated cyanurates (H2ClCy) was estimated at 0.5 x 10(5) M(-1) s(-1). In contrast, the rates with the NH2Cl solution containing H3Cy increased with an increase in H3Cy concentrations, increasing from 1.2 x 10 to 2.7 x 10 M(-1) s(-1) by the addition of 1.55 mM H3Cy. The DPD color development rates (OD512/t1/2/M) with free available chlorine (0.015mM) declined from 1.3 x 10(5) to 0.9 x 10(5)M(-1) by the addition of 0.61 mM H3Cy.