Dioxin suppresses benzo[a]pyrene-induced mutations and DNA adduct formation through cytochrome P450 1A1 induction and (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide inactivation in human hepatoma cells

Benzo[a]pyrene (BaP) is metabolically activated by cytochrome P450 enzymes, and forms DNA adduct leading to mutations. Cytochrome P450 1A1 plays a central role in this activation step, and this enzyme is strongly induced by chemical agents that bind to the aryl hydrocarbon receptor (AhR), which is also known as a dioxin receptor. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR ligand has not been shown to form any DNA adduct, but has a possibility to aggravate the toxicity of precarcinogenic polycyclic hydrocarbons through the induction of metabolic enzymes. We treated human hepatoma cells (HepG2) with TCDD, and subsequently exposed them to BaP to elucidate the synergistic effects on mutations. Surprisingly, mutant frequency induced by BaP at the hypoxanthine-guanine phosphribosyltransferase (HPRT) locus was decreased by pretreatment with TCDD. In correlation with decrease in the mutant frequencies, BaP–DNA adduct formation was also decreased by TCDD pretreatment. This suppressive effect of TCDD was more potent when the cells were exposed to (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), a reactive metabolic intermediate of BaP. Among the enzymes catalyzing BaP oxidation and conjugation, cytochrome P450 1A1, 1A2, 3A4 and UDP-glucuronosyltransferase 1A1 mRNAs were induced by the exposure to TCDD. In cytochrome P450 1A1-deficient murine cells and cytochrome P450 1A1-uninducible human cells, TCDD could not suppress BPDE–DNA adduct formation. Further experiments using “Tet-On” cytochrome P450 1A1-overexpressing cells and a recombinant cytochrome P450 1A1 enzyme demonstrated that this is the key enzyme involved in the biotransformation of BaP, that is, both production and inactivation of BPDE. We conclude that TCDD-induced cytochrome P450 catalyzes the metabolism of BPDE to as yet-unidentified products that are not apparently DNA-reactive, thereby reducing mutations in hepatoma cells.     

Rates of Serious Intracellular Infections in Autoimmune Disease Patients Receiving Initial Glucocorticoid Therapy

Background/Aims

The Japanese National Hospital Organization evidence-based medicine (EBM) Study group for Adverse effects of Corticosteroid therapy (J-NHOSAC) is a Japanese hospital-based cohort study investigating the safety of the initial use of glucocorticoids (GCs) in patients with newly diagnosed autoimmune diseases. Using the J-NHOSAC registry, the purpose of this observational study is to analyse the rates, characteristics and associated risk factors of intracellular infections in patients with newly diagnosed autoimmune diseases who were initially treated with GCs.

Methodology/Principal Findings

A total 604 patients with newly diagnosed autoimmune diseases treated with GCs were enrolled in this registry between April 2007 and March 2009. Cox proportional-hazards regression was used to determine independent risk factors for serious intracellular infections with covariates including sex, age, co-morbidity, laboratory data, use of immunosuppressants and dose of GCs. Survival was analysed according to the Kaplan-Meier method and was assessed by the log-rank test. There were 127 serious infections, including 43 intracellular infections, during 1105.8 patient-years of follow-up. The 43 serious intracellular infections resulted in 8 deaths. After adjustment for covariates, diabetes (Odds ratio [OR]: 2.5, 95% confidence interval [95% CI] 1.1–5.9), lymphocytopenia (≦1000/μl, OR: 2.5, 95% CI 1.2–5.2) and use of high-dose (≧30 mg/day) GCs (OR: 2.4, 95% CI 1.1–5.3) increased the risk of intracellular infections. Survival curves showed lower intracellular infection-free survival rate in patients with diabetes, lymphocytopaenia and high-dose GCs treatments.

Conclusions/Significance

Patients with newly diagnosed autoimmune diseases were at high risk of developing intracellular infection during initial treatment with GCs. Our findings provide background data on the risk of intracellular infections of patients with autoimmune diseases. Clinicians showed remain vigilant for intracellular infections in patients with autoimmune diseases who are treated with GCs.     

Measuring Airway Surface Liquid Depth in Ex Vivo Mouse Airways by X-Ray Imaging for the Assessment of Cystic Fibrosis Airway Therapies

In the airways of those with cystic fibrosis (CF), the leading pathophysiological hypothesis is that an ion channel defect results in a relative decrease in airway surface liquid (ASL) volume, producing thick and sticky mucus that facilitates the establishment and progression of early fatal lung disease. This hypothesis predicts that any successful CF airway treatment for this fundamental channel defect should increase the ASL volume, but up until now there has been no method of measuring this volume that would be compatible with in vivo monitoring. In order to accurately monitor the volume of the ASL, we have developed a new x-ray phase contrast imaging method that utilizes a highly attenuating reference grid. In this study we used this imaging method to examine the effect of a current clinical CF treatment, aerosolized hypertonic saline, on ASL depth in ex vivo normal mouse tracheas, as the first step towards non-invasive in vivo ASL imaging. The ex vivo tracheas were treated with hypertonic saline, isotonic saline or no treatment using a nebuliser integrated within a small animal ventilator circuit. Those tracheas exposed to hypertonic saline showed a transient increase in the ASL depth, which continued for nine minutes post-treatment, before returning to baseline by twelve minutes. These findings are consistent with existing measurements on epithelial cell cultures, and therefore suggest promise for the future development of in vivo testing of treatments. Our grid-based imaging technique measures the ASL depth with micron resolution, and can directly observe the effect of treatments expected to increase ASL depth, prior to any changes in overall lung health. The ability to non-invasively observe micron changes in the airway surface, particularly if achieved in an in vivo setting, may have potential in pre-clinical research designed to bring new treatments for CF and other airway diseases to clinical trials.     

A Japanese child with geleophysic dysplasia caused by a novel mutation of FBN1

Geleophysic dysplasia (GD) is a rare disorder characterized by severe short stature, short hands and feet, limited joint mobility, skin thickening, characteristic facial features (e.g., a “happy” face), and cardiac valvular disorders that often result in an early death. The genes ADAMTSL2 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif-like 2) and FBN1 (fibrillin 1) were recently identified as causative genes for GD. Here, we describe a 10-year-old Japanese female with GD who was born to non-consanguineous parents. At the age of 11 months, she was referred to our hospital because of very short stature for her age (− 4.4 standard deviations of the age-matched value) and a “happy” face with full cheeks, a shortened nose, hypertelorism, and a long and flat philtrum, characteristic of GD. Her hands and feet were small, her skin was thickened, and her joint mobility was generally limited. She had cardiac valvular disorders and history of recurrent respiratory failure. Mutation analysis revealed no abnormalities in ADAMTSL2. However, analysis of FBN1 revealed a novel heterozygous mutation (c.5161T > T/G) in exon 41, which encodes transforming growth factor-β-binding protein-like domain 5 (TB5). GD is an extremely rare disorder and, to our knowledge, only one case of GD with an FBN1 mutation has been reported in Japan. Similar to the previously reported cases of GD, the mutation in the current patient was located in the TB5 domain, which suggests that abnormalities in this domain of FBN1 are responsible for GD.     

Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.     

Topogenesis and Homeostasis of Fatty Acyl-CoA Reductase 1

Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.     

Adipogenetic effects of retrofractamide A derivatives in 3T3-L1 cells

In a previous study, retrofractamide A from the fruit of Piper chaba was shown to promote adipogenesis in 3T3-L1 cells. In the present study, retrofractamide A and its derivatives were synthesized, and their adipogenetic effects in 3T3-L1 cells were examined. Among the tested compounds, an amide composed of 9-(3′,4′-methylenedioxyphenyl)-nona-2E,4E,8E-trienoic acid and an n-butyl or n-pentyl amine showed strongest activity. Moreover, the amide with the n-pentyl amine moiety significantly increased the uptake of 2-deoxyglucose into the cells, and also increased the mRNA levels of adiponectin, peroxisome proliferator-activated receptor γ2 (PPARγ2), glucose transporter 4 (GLUT4), fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein (C/EBP) α and β in a similar manner as the PPARγ agonist troglitazone, although it had less agonistic activity against PPARγ.     

Chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy

We have developed chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy. Chemically modified RNAs possessing urea/thiourea-bridged aromatic compounds instead of naturally occurring dinucleotides at the 3′-overhang region were easily prepared in good yields and were more resistant to nucleolytic hydrolysis than unmodified RNA. siRNAs containing urea or thiourea derivatives showed the desired knockdown effect. Furthermore, modified miR-143 duplexes carrying the urea/thiourea compounds in the 3′-end of each strand were able to inhibit the growth of human bladder cancer T24 cells.     

Three-Dimensional Protein Fold Determination from Backbone Amide Pseudocontact Shifts Generated by Lanthanide Tags at Multiple Sites

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Anti-cancer effects of naturally occurring compounds through modulation of signal transduction and miRNA expression in human colon cancer cells

Much evidence indicates that various naturally occurring compounds have an anti-cancer effect, but the detailed mechanisms are not well understood. In this study, we selected anti-cancer phytochemicals such as epigallocatechin-3-gallate (EGCG), resveratrol (RES) and α-mangostin (α-M), all of which are well-characterized chemopreventive agents. We sought to elucidate the mechanism of their anti-cancer effects and the synergistic effects obtained by combined treatment with the anti-cancer drug 5-fluorouracil (5-FU) in three human colon cancer cell lines. The numbers of viable cells were consistently decreased by the treatment with EGCG, RES or α-M at more than 10 μM in all three cell lines tested. All compounds mainly induced apoptosis and suppressed the PI3K/Akt signaling pathway. Additionally, α-M, which had the greatest PI3K/Akt-suppressing activity, also suppressed MAP kinase (MAPK)/Erk1/2 signaling. Importantly, the combination treatment with RES and 5-FU induced a remarkably synergistic enhancement of growth inhibition and apoptosis through the additional suppression of the MAPK/Erk1/2 signaling pathway in colon cancer DLD-1 cells. Interestingly, RES increased the intracellular expression level of miR-34a, which down-regulated the target gene E2F3 and its downstream Sirt1, resulting in growth inhibition. These findings indicate that these compounds functioned as chemosensitizers when combined with anti-cancer drugs through the modulation of apoptotic and growth-related signaling pathways. Also, RES exerted its anti-cancer activity in part through a newly defined mechanism, i.e., the miR-34a/E2F3/Sirt1 cascade.