Ryushi Nozawa

Estrogenic activity of alkylphenols, bisphenol S, and their chlorinated derivatives using a GFP expression system

Alkylphenol ethoxylates, widely used non-ionic surfactants, are biodegraded into alkylphenols such as nonylphenol (NP) and t-octylphenol (OP), short-chain ethoxylates such as NP-monoethoxylate (NP1EO) and NP-diethoxylate (NP2EO), and alkylphenoxy carboxylic acids such as 4-t-octylphenoxyacetic acid (OP1EC). Bisphenol S (BPS) is more heat-stable and photo-resistant than bisphenol A (BPA), and therefore replaces BPA. These chemicals could be chlorinated during wastewater treatment. We synthesized these compounds and their chlorinated derivatives to estimate their estrogenic activities using a GFP expression system. The EC(50) ranking of NP-related compounds was NP > ClNP > diClNP > NP1EO > ClNP1EO > NP2EO. The estrogenic activity of OP1EC was 10 times less potent than parent OP. Furthermore, BPS showed comparable estrogenic activity with BPA. The EC(50) ranking of BPS-related compounds was BPA ≥ BPS > triClBPS > diClBPS > ClBPS. Other tested BPS derivatives had no estrogenic activity. Chlorination of the tested chemicals did not enhance their estrogenic activity, in contrast to certain chlorinated BPAs. Thus, our results demonstrated that chlorinated derivatives of NP, OP, and BPS, even if artificially produced during wastewater processing, were less estrogenic than their parent chemicals, known as endocrine disruptors.

S100A8 and S100A9 Overexpression Is Associated with Poor Pathological Parameters in Invasive Ductal Carcinoma of the Breast

S100 protein A8 and A9 naturally form a stable heterocomplex. Recently, we have proved that S100A9 overexpression in various adenocarcinomas is associated with poor tumor differentiation. In this study, we examined the relationship between the expression of each protein and the pathological parameters that reflect the aggressiveness of carcinoma, in invasive ductal carcinoma (IDC) of the breast. Serial paraffin-embedded tissue sections from 101 IDC cases were immunostained with respective monoclonal antibodies, and the results were as follows: 1) A positive correlation of immunoreactivity between S100A8 and S100A9 was noticed (r=0.873 and P<0.0001); 2) The percentage of S100A9-positive tumor cells was higher than that of S100A8-positive tumor cells (P<0.001), and S100A8 alone was not detected in any case; 3) Overlap between S100A8 and S100A9 staining patterns was found in the corresponding tissue areas, but S100A9 positivity was also observed in S100A8-negative tumor cells; 4) The immunopositivity for each protein also correlated with the mitotic activity, MIB-1 index, HER2 overexpression, node metastasis, and poor pT categories and pStage (P<0.05); 5) Co-expression of both proteins was associated with poor tumor differentiation, vessel invasion, node metastasis, and poor pStage (P<0.05). Furthermore, co-expression of the proteins was also observed in MCF-7 cells, and it was suggested that the immunolocalization is related with cell cycle. Our conclusions are as follows: 1) It is suggested that S100A8 is S100A9-dependently expressed and acquires the protein stability by S100A8/A9 heterocomplex formation; 2) S100A8 and S100A9 overexpression should be considered marker of poor prognosis in IDC.

DPPH Radical-Scavenging Compounds from Dou-Chi, a Soybean Fermented Food

Dou-chi, a traditional soybean food fermented with Aspergillus sp., is usually used as a seasoning in Chinese food, and has also been used as a folk medicine in China and Taiwan. As 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavengers, four phenol compounds, one isoflavanone, eight isoflavones and one 4-pyrone have been isolated from dou-chi. Among these fourteen compounds, 3′-hydroxydaidzein, dihydrodaidzein and a 4-pyrone compound have not yet been isolated from soybean miso. The structure of the novel 4-pyrone compound, 3-((E)-2-carboxyethenyl)-5-(4-hydroxyphenyl)-4-pyrone-2-carboxylic acid was elucidated by using the same compound as that obtained from the biotransformation of daidzein. 3′-Hydroxydaidzein showed as high DPPH radical-scavenging activity as that of α-tocopherol, and 6-hydroxydaidzein had mushroom tyrosinase inhibitory activity with an IC50 value of 10 μM. The order of estrogenic activity is as follows: genistein > daidzein >> 3′-hydroxydaidzein > 8-hydroxygenistein, using a green fluorescent protein expression system. Furthermore, the contents of isoflavones in the fermentation process of dou-chi were measured.

Evaluation of S100A10, annexin II and B‐FABP expression as markers for renal cell carcinoma

This study aimed to analyze expression of S100A10, annexin II and B‐FABP genes in renal cell carcinoma (RCC) and their potential value as tumor markers. Furthermore, any correlation between the gene expression and prognostic indicators of RCC was analyzed. Expression of each gene was estimated by RT‐PCR in the non‐neoplastic (normal) and tumorous parts of resected kidney samples. Also, each antigen was immunostained in RCC and normal kidney tissues. Expression of the S100A10 gene averaged 2.5‐fold higher in the tumor than that in the normal tissues (n = 47), after standardization against that of β‐actin. However, expression of annexin II, a natural ligand of S100A10, was only 1.64‐fold higher. In the tissue sections of RCC, S100A10 and annexin II were immunostained in membranes. In the normal renal epithelia, however, both antigens were stained in the Bowmans capsule and the tubules from Henles loop through the collecting duct system, but not in the proximal tubules, from where most RCC are derived. In contrast, expression of the B‐FABP gene was 20‐fold higher in the tumor. No B‐FABP was immunohistochemically detected in normal kidney sections, but it was stained in the cytoplasm of RCC tissue sections. S100A10 and B‐FABP genes were overexpressed regardless of nuclear grade and stage of RCC. Immunopositivity in RCC tissues (n = 13) was 100% for S100A10 and annexin II, and 70% for B‐FABP; however, no clear relationship was observed in either antigen with nuclear grade and stage. It was found that all three performed well as RCC markers. B‐FABP was most specific to RCC, as it was expressed little in normal kidney tissues. (Cancer Sci 2007; 98: 77–82)

Immunohistochemical investigation of migration inhibitory factor-related protein (MRP)-14 expression in hepatocellular carcinoma

Migration inhibitory factor-related protein (MRP)-8 and-14 belong to the S-100 protein family and are associated with myeloid cell differentiation. MRP is also expressed in some epithelia. However, there are few reports for the investigation on carcinomas.Using the monoclonal antibody 60B8 against MRP-14, we carried out the immunohistochemical evaluation of MRP-14 expression in 70 cases of hepatocellular carcinoma (HCC), and examined the relation to tumor differentiation and vascular invasion. Positively stained tumor cells were detected in 32 cases, all of which belonged to grade II (7/30) or grade III (25/25) of the Edmondson-Steiner classification. In particular, the grade III HCC showed a significantly greater positive reaction. Immunopositivity in the non-carcinomatous hepatocytes and bile duct epithelia was not observed.These findings suggested that malignant hepatocytes newly express MRP-14 and that the neo-expression in differentiated HCC is related to the tumor differentiation and shows higher correlation in the poorly differentiated carcinomas. Furthermore, the cholangiocellular carcinoma and metastatic adenocarcinoma as control materials also presented a more marked immunoreactivity for MRP-14 in the poorly differentiated carcinomas, in a similar manner with the findings of the HCC. Accordingly, MRP is considered to be frequently neo-expressed in poorly differentiated carcinomas.MRP-14 expression rate in the 48 HCC cases with vascular invasion was 56%, showing no significant difference compared with non-invasive tumors.

Seroprevalence of Helicobacter pylori Infection in Patients with Connective Tissue Diseases

To assess the possibility that Helicobacter pylori might be an etiologic agent, titers of anti‐H. pylori IgG in sera of patients with connective tissue diseases [rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyositis or dermatomyositis (PM/DM), progressive systemic sclerosis (PSS), mixed connective tissue disease (MCTD) and Sjögrens syndrome (SjS)] were compared with those of non‐patient (healthy) volunteers and of patients with chronic pulmonary diseases (CPD) by ELISA using an extract of sonicated H. pylori as the antigen. Among patients with connective tissue diseases, those with SLE and RA had anti‐H. pylori titers as low as healthy volunteers. Patients with SjS had much higher average titers than patients with CPD (P<0.05). We previously reported that levels of myeloid calcium‐binding protein (MRP8 and MRP14) were elevated in the serum of patients with connective tissue diseases. No correlation was found between serum levels of anti‐H. pylori IgG and of MRP, a novel marker of inflammation. Furthermore, sera with high IgG titers were selected, and their reactivity with the H. pylori antigen were analyzed by Western blotting. H. pylori antigens with a variety of molecular masses were immunostained with sera from patients and from healthy volunteers, but a 16‐kDa antigen was only immunostained by reaction with the sera of patients with MCTD and SjS, although the number of test samples was small.

Inhibition by the protein kinase inhibitor HA1077 of the activation of NADPH oxidase in human neutrophils

The effect of an inhibitor of protein kinase, HA1077 [1-(5-isoquinolinesulfonyl)-homopiperazine HCl], and its hydroxylated metabolite, HA1100, on the activation of NADPH oxidase in human neutrophils were studied. Cells were preincubated with each drug for 10 min and then activated by treatment with phorbol myristate acetate (PMA) or formylmethionyl leucyl phenylalanine (FMLP). After activation, the rate of superoxide dismutase-inhibitable reduction of cytochrome c was estimated. HA1077 and HA1100 inhibited the PMA-induced production of O2- by neutrophil NADPH oxidase in a concentration-dependent manner (IC50 = 15 and 24 microM, respectively). The sensitivity of the FMLP-induced production of O2- to these drugs was similar. The production of O2- in 1,25-dihydroxyvitamin D3-treated HL-60 cells, which differentiated to macrophage-like cells, was also inhibited by the drugs. The extent of inhibition by HA1077 was almost the same as that by a calmodulin inhibitor (W-7) and by inhibitors of protein kinase (H-7 and H-8). In a cell-free lysate of neutrophils, the NADPH-dependent production of O2- can be induced by sodium dodecyl sulfate (SDS). HA1077 at 100 microM had only a weak inhibitory effect on the cell-free, SDS-induced production of O2-, an indication that HA1077 inhibits the activation of NADPH oxidase, not the actual activity. The effects of H-7 and H-8 were similar to that of HA1077, whereas W-7 inhibited the production of O2- by the cell-free extract of HL-60 cells. This action of HA1077 could explain, in part, its ability to protect neuronal cells from death after ischemia.

Characterization of regulatory sequences and nuclear factors that function in cooperation with the promoter of the human thymidylate synthase gene

To identify the essential sequence of the promoter of the human thymidylate synthase (hTS) gene, deletion mutants were constructed and assayed for promoter activity. The essential sequence was located within 65 bp upstream from the major cap site and a sequence that reduces the promoter activity was found in a region upstream from the essential promoter sequence. We previously identified two DNA-binding nuclear factors, NF-TS2 and NF-TS3, that bind to a region around the site of initiation of translation of the hTS gene. In this study, we confirmed the binding site of these factors by gel mobility shift analysis and found that NF-TS2 is the major factor that binds to the hTS gene in HeLa cells, whereas NF-TS3 is the major factor in the TIG-1 line of human fibroblast cells. To clarify the function of these factors, we examined the effects of the binding of these factors on the promoter activity. Our findings suggest that the binding of NF-TS2 enhances the promoter activity of the hTS gene in HeLa cells, whereas the binding of NF-TS3 represses the activity of the same promoter in TIG-1 cells.

Susceptibility of Mice to Bacterial and Fungal Infections after Intragastric Administration of Ebselen

The seleno‐organic compound ebselen (2−phenyl−1,2−benzisoselenazol−3(2H)‐one) has anti‐inflammatory activity and exhibits glutathione peroxidase‐like activity in‐vitro. Ebselen inhibited candidacidal activity over the same range of concentrations as it inhibited the production of microbicidal H2O2 by human neutrophils and macrophage‐like cells. Therefore, the long‐term administration of ebselen might be expected to induce an immunocompromised state in the host. To examine such a possibility, mice (5−weeks‐old ddY, male) were given daily intragastric doses of 0, 10 or 100 mg kg−1 ebselen for 21 days and then infected intraperitoneally with Candida albicans (108 cells/mouse), Pseudomonas aeruginosa (1.5 times 107 cells/mouse) or methicillin‐resistant Staphylococcus aureus (5 times 108 cells/mouse). Ebselen at none of the tested doses affected the increase in body weight of mice during administration of the drug. No evidence was obtained that mice became more susceptible to the various microorganisms after the administration of ebselen at any tested dose.