Effects of bezafibrate, PPAR pan-agonist, and GW501516, PPARdelta agonist, on development of steatohepatitis in mice fed a methionine- and choline-deficient diet

We evaluated the effects of bezafibrate, a peroxisome proliferator-activated receptor (PPAR) pan-agonist, and GW501516, a PPARdelta agonist, on mice fed a methionine- and choline-deficient (MCD) diet, a model of non-alcholic steatohepatitis (NASH), to investigate (a) the efficacy of bezafibrate against non-alcholic steatohepatitis and (b) the relation between non-alcholic steatohepatitis and the functional role of PPARdelta. Bezafibrate (50 or 100 mg/kg/day) and GW501516 (10 mg/kg/day) were administered by gavage once a day for 5 weeks. Hepatic lipid contents, plasma triglyceride, high density lipoprotein (HDL)-cholesterol and alanine aminotransferase (ALT) concentrations were evaluated, as were histopathological changes in the liver and hepatic mRNA expression levels. Bezafibrate and GW501516 inhibited the MCD-diet-induced elevations of hepatic triglyceride and thiobarbituric acid-reactants contents and the histopathological increases in fatty droplets within hepatocytes, liver inflammation and number of activated hepatic stellate cells. In this model, bezafibrate and GW501516 increased the levels of hepatic mRNAs associated with fatty acid beta-oxidation [acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-1 (CPT-1), liver-fatty acid binding protein (L-FABP) and peroxisomal ketothiolase], and reduced the levels of those associated with inflammatory cytokines or chemokine [transforming growth factor (TGF)-beta1, interleukin (IL)-6, IL-1beta, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) alpha and nuclear factor (NF)-kappaB1]. In addition, bezafibrate characteristically reduced the elevation in the level of plasma ALT, but enhanced that in plasma adiponectin and increased the mRNA expression levels of its receptors (adiponectin receptors 1 and 2). These results suggest that (a) bezafibrate (especially) and GW501516 might improve hepatic steatosis via an improvement in fatty acid beta-oxidation and a direct prevention of inflammation, (b) treatment with a PPARdelta agonist might improve non-alcholic steatohepatitis, (c) bezafibrate may improve non-alcholic steatohepatitis via activation not only of PPARalpha but also of PPARdelta, because bezafibrate is a PPAR pan-agonist.     

Inhibition of autophosphorylation of epidermal growth factor receptor by small peptides in vitro

1. Inhibition of uncontrolled epidermal growth factor receptor (EGFR) is one of the approaches for the treatment of breast and lung cancers. We designed oligopeptides consisting of amino-acid sequences of the major (Y1068, Y1148, and Y1173) and minor (Y992) autophosphorylation sites of EGFR. These peptides may be exogenous substrates or pseudosubstrates that interfere with the autophosphorylation of EGFR. The effects of the peptides on autophosphorylation of EGFR were studied. 2. Purified EGFR was phosphorylated in vitro with EGF in the presence of various synthetic peptides. The phosphorylation level of EGFR was then evaluated after SDS-PAGE separation, followed by Western blot analysis with antiphosphotyrosine antibody. 3. Ac-VPEYINQ-NH2 (Y1068) and Ac-DYQQD-NH2 (Y1148) showed the most potent inhibitory effects, followed by Ac-ENAEYLR-NH2 (Y1173). These peptides at 4 mM suppressed phosphorylation to 30-50%. 4. Combination of the three kinds of peptides much more strongly inhibited autophosphorylation. The 50% inhibitory concentration (IC50) value was 0.5 mM as a mixture and was comparable to that of AG1478 (IC50, 0.3 mM) at 0.2 mM ATP. 5. Neither Ac-DIYET-NH2 or Ac-KIYEK-NH2, designed previously based on the amino-acid sequence of an autophosphorylation site of insulin receptor, nor their related (Ac-KIFMK-NH2) or unrelated (Ac-LPFFD-NH2) peptides showed an inhibitory effect. These results suggest that the small peptides that originated from the autophosphorylation sites of EGFR interact solely with EGFR. 6. The peptides containing the sequences surrounding Y1068, Y1148, and Y1173 may be a promising seed for the development of therapeutic agents for breast and lung cancers.     

Synthesis and antituberculosis activity of a novel series of optically active 6-nitro-2,3-dihydroimidazo[2,1-b]oxazoles

In an effort to develop potent new antituberculosis agents that would be effective against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis, we prepared a novel series of optically active 6-nitro-2,3-dihydroimidazo[2,1-b]oxazoles substituted at the 2-position with various phenoxymethyl groups and a methyl group and investigated the in vitro and in vivo activity of these compounds. Several of these derivatives showed potent in vitro and in vivo activity, and compound 19 (OPC-67683) in particular displayed excellent in vitro activity against both drug-susceptible and drug-resistant strains of M. tuberculosis H37Rv (MIC = 0.006 microg/mL) and dose-dependent and significant in vivo efficacy at lower oral doses than rifampicin in mouse models infected with M. tuberculosis Kurono. The synthesis and structure-activity relationships of these new compounds are presented.     

Pentavalent vanadium induces hepatic metallothionein through interleukin-6-dependent and -independent mechanisms

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein which has a high affinity for metals. The synthesis of MT is induced by heavy metals such as cadmium and zinc. However, little is known about the induction of MT by tetravalent or pentavalent metals. We investigated the induction of MT synthesis by a pentavalent vanadium compound in mice. Hepatic MT concentrations were increased by subcutaneous injection of ammonium metavanadate (AMV) dose-dependently, and to the similar levels as those induced by zinc chloride. However, accumulation of vanadium in the liver was very low, while high concentrations of vanadium were detected in the kidney. High performance liquid chromatography/inductively coupled argon plasma-mass spectrometry (HPLC/ICP-MS) chromatogram of the liver cytosol of AMV-treated mice revealed that the major metal bound to MT was not vanadium, but zinc. The chromatogram of the liver cytosol of MT null mice demonstrated the existence of a low-molecular-weight vanadium-binding protein that is different from MT. A time-course study showed that concentrations of serum interleukin-6 (IL-6) and serum amyloid A (SAA), an acute-phase protein, increased after the AMV injection. To confirm the involvement of IL-6 in MT induction by AMV administration, IL-6 null and wild-type mice were injected with AMV. In IL-6 null mice, hepatic MT induction by AMV administration decreased significantly to about a half of wild-type mice. These data suggest that both IL-6-dependent and -independent mechanisms are involved in MT induction by vanadium compounds in mice.     

Induction of lupus-related specific autoantibodies by non-specific inflammation caused by an intraperitoneal injection of n-hexadecane in BALB/c mice

A single intraperitoneal (i.p.) injection of pristane, incomplete Freund's adjuvant (IFA), or the adjuvant oil squalene, but not high molecular weight medicinal mineral oils, induces lupus-related autoantibodies to nRNP/Sm and -Su in non-autoimmune strains of mice. This ability appears to be associated with the low molecular weight and adjuvanticity of hydrocarbon. n-Hexadecane (C(16)H(34)), which is present in petroleum, has adjuvant activity and induces arthritis in rodents like other lupus-inducing oils. In addition to dietary exposure to n-hexadecane in mineral oils, exposure also occurs via inhalation of oil mist, jet fuel, or diesel exhaust or by absorption through the skin. Since n-hexadecane is a low molecular weight adjuvant hydrocarbon oil similar to other lupus-inducing hydrocarbons, the present study examined whether it can also induce lupus-related autoantibodies in mice. Female BALB/cJ mice received a single i.p. injection of 0.5 ml of n-hexadecane, pristane, or saline (control). Pathology and serology (immunoglobulin levels, autoantibodies by immunofluorescence, immunoprecipitation, and ELISA) were examined 3 months later. Unexpectedly, all n-hexadecane-treated mice, but none in the other groups, developed inflammatory ascites within 2.5 months. n-Hexadecane induced hypergammaglobulinemia (IgG1, IgG2a), antinuclear (titer>1:160, 67%) and -cytoplasmic antibodies (58%) and autoantibodies to nRNP/Sm (25%), Su (33%), ssDNA (83%), and chromatin (100%). Therefore, non-specific inflammation caused by n-hexadecane resulted in the production of a limited set of specific autoantibodies. These previously unrecognized immunological effects of n-hexadecane may have implications in monitoring human exposure to hydrocarbons and in the pathogenesis of autoimmune diseases.     

Visual display terminal work and sick building syndrome--the role of psychosocial distress in the relationship

The present study investigated the association between visual display terminal (VDT) work and sick building syndrome (SBS) and the role of psychosocial factors in the relationship. Subjects were 2,161 Japanese office workers who responded to a cross-sectional anonymous self-administered questionnaire survey. Questions included were derived from the Milj?medicin 040, a validated questionnaire on SBS symptoms. After exclusion of data with missing information, data for 1,881 subjects were used for analysis. Multivariate logistic regression was used to estimate the odds ratio for SBS with adjustment for potential confounding factors, including psychosocial work stress. In multivariate analysis, the odds ratio for SBS was significantly elevated for men engaged in VDT work for 4 or more hours a day (OR=2.5, 95%CI: 1.0, 5.9) compared with less than 1 hour a day, showing a significant trend association (P for trend=0.04). In women, although the odds ratio for SBS with VDT use of 4 or more hours a day was somewhat elevated with adjustment for non-psychosocial factors (OR=1.5, 95%CI: 0.5, 4.3), the increase was greatly attenuated after adjustment for psychosocial work distress (OR=1.1). In conclusion, our study suggests that extended hours of VDT use might be related to increased SBS symptoms. Moreover, psychosocial distress related to VDT work might mediate the relationship between VDT use and SBS symptoms in women.     

Application of aldehyde dehydrogenase 2 (ALDH2) genetic diagnosis in support of decreasing alcohol intake

Encouraging behavioral changes to decrease alcohol intake is not easy from the standpoint of health support. This study was conducted to examine whether the genetic diagnosis of ALDH2 polymorphism is useful in supporting those who want to decrease their alcohol intake. The participants in this study were 329 male employees who wanted to know the result of their ALDH2 genotype. We divided the 329 participants randomly into two groups. One was the "notified group" (n=157), and the other was the "non-notified group" (n=172). The subjects belonging to the "notified group" were informed of the results of the ALDH2 genotype diagnosis in April, 2003. Drinking habits and laboratory data were obtained before and after notification of the ALDH2 genotype. Among those with genotype ALDH2*1/*1, there was no significant change in drinking frequencies, nor was there any significant decline in liver function laboratory data in either of the groups before and after notification of the genotype. However, weekly alcohol intake tended to increase compared to that before notification. On the other hand, with regard to those with genotype ALDH2*1/*2, no significant changes in drinking frequencies or liver function laboratory data were evident in either group before and after notification of the genotype. However, the weekly alcohol intake tended to increase in the non-notified group, whereas it tended to decrease in the notified group. Although the result was not significant, it is suggested that, with further study and an increased sample size, the genetic diagnosis may be found to be useful.     

hOGG1 Ser326Cys polymorphism and risk of hepatocellular carcinoma among Japanese

BACKGROUND: The Ser326Cys polymorphism in human oxoguanine glycosylase 1 (hOGG1), which is involved in the repair of 8-hydroxy-2-deoxyguanine in oxidatively damaged DNA, has been associated with susceptibility to certain cancers, but has not been examined in causation of hepatocellular carcinoma (HCC). METHODS: We conducted a case-control study to investigate whether this polymorphism was related to HCC risk with any interaction with alcohol consumption and cigarette smoking. Genotyping was performed by a polymerase chain reaction with confronting two-pair primers among 209 newly diagnosed HCC cases, 275 hospital controls, and 381 patients with chronic liver disease (CLD) without HCC. RESULTS: Overall, the hOGG1 genotype was not significantly associated with HCC; adjusted odds ratios (and 95% confidence intervals) for the Ser/Cys and Cys/Cys genotypes compared with the Ser/Ser genotype were 0.79 (0.35-1.79) and 0.48 (0.18-1.27) against hospital controls, and 1.51 (0.96-3.37) and 0.86 (0.50-1.47) against CLD patients. We could not detect any significant gene-alcohol interaction (p = 0.95 or 0.16) or gene-smoking interaction (p = 0.70 or 0.69). CONCLUSIONS: These results suggest that the hOGG1 Ser326Cys polymorphism may not play a major role as an independent factor in hepatocarcinogenesis.     

KmrA multidrug efflux pump from Klebsiella pneumoniae

We cloned a gene responsible for multidrug resistance from the chromosomal DNA of Klebsiella pneumoniae MGH78578 that showed multidrug resistance. We designated the gene kmrA. The deduced amino acid sequence of KmrA was similar to that of SmvA that is responsible for methyl viologen-resistance in Salmonella enterica sv. Typhi and Typhimurium. Introduction of the cloned kmrA gene into drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33342, tetraphenylphosphonium chloride (TPPCl), methyl viologen and ethidium bromide. We observed elevated energy-dependent efflux of ethidium in E. coli cells carrying the kmrA gene compared with control cells. We also cloned the smvA gene from S. enterica sv. Typhimurium LT2 and investigated the resistance pattern for several drugs. The pattern was similar between KmrA from K. pneumoniae and SmvA from S. enterica.