A new luciferase reporter gene assay for the detection of androgenic and antiandrogenic effects based on a human prostate specific antigen promoter and PC3/AR human prostate cancer cells.

We developed a new mammalian cell-based luciferase reporter gene assay for androgenic and antiandrogenic activities of chemicals and environmental samples. Environmental samples usually have a complex matrix that may contain the constituents acting as androgen receptor (AR) agonists, AR antagonists or aryl hydrocarbon receptor (AhR) agonists. AhR agonists are known to elicit the antiandrogenic effect through cross-talk between AR and AhR signal transduction pathways. In this study, PC3/AR human prostate carcinoma cells were transiently transfected with a prostate-specific antigen (PSA) promoter-driven luciferase expression plasmid. The cells were treated with a test compound or an environmental sample for 24 h at 37 degrees C and then measured for luciferase activity. The luciferase activity was induced by dihydrotestosterone (DHT) in a concentration-dependent manner in a concentration range from 10 fM to 1 nM. R1881, a synthetic androgen receptor agonist, induced luciferase activity and its inductive effects was additive to that of DHT. The luciferase activity was not induced by cortisol, a glucocorticoid, progesterone, a progestin, and 17beta-estradiol, an estrogen in a concentration range of up to 1 microM. DHT-induced luciferase activity was reduced by bicalutamide and cyproterone acetate, AR antagonists, and also by benzo[a]pyrene, an aryl hydrocarbon receptor agonist, through AhR-mediated pathways. All of these findings indicate that the present assay system correctly responds to AR agonists, AR antagonists and AhR agonist and, therefore, it is a powerful tool for the sensitive and selective screening of chemicals and environmental samples for their androgenic and antiandrogenic activities. We developed the first assay system, in which the expression of luciferase was driven by the promoter of a prostate-specific antigen gene, a typical human androgen-regulated gene.     

Hair analysis of nicotine and cotinine for evaluating tobacco smoke exposure by liquid chromatography-mass spectrometry

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method for the determination of nicotine and cotinine in human hair was established. In the procedure, a hair sample (10 mg) was washed with dichloromethane and digested in 2.5 M sodium hydroxide. The digest was extracted with dichloromethane and then 25 mM hydrochloric acid in methanol was added to the extract, to prevent loss of analytes. The solution was evaporated and redissolved in the mobile phase, methanol/10 mM ammonium acetate (30/70, v/v). A 20 microL aliquot of redissolved solution was subjected to analysis. Nicotine and cotinine in human hair were quantified by using deuterated analytes as internal standards. The quantification limits were 8 microg/L for nicotine and 0.9 microg/L for cotinine. The proposed method was applied to measure the concentrations of nicotine and cotinine in hair of smokers and non-smokers to evaluate their self-reported smoking and exposure to environmental tobacco smoke. In both cases, the method provided good selectivity, accuracy and precision.     

Hydrogen Effect on Coke Removal and Catalytic Performance in Pre-Carburization and Methane Dehydro-Aromatization Reaction on Mo/HZSM-5

1. Introduction As an effective utilization of methane, the methane dehydro-aromatization was focused in the last decade [1-28]. Over the Mo/HZSM-5 bi- functional catalyst at high reaction temperature, methane can be converted into light aromatics (ben- zene and naphthalene) and hydrogen. Mo active species can activate the C—H bond of methane; and HZSM-5 supplies the acid sites for the oligomeriza- tion and cyclization of hydrocarbons to form aromat- ics, and suppresses the deeper condens…     

Mediated amperometric biosensor for hypoxanthine based on a hydroxymethylferrocene-modified carbon paste electrode

An amperometric enzyme electrode for the determination of hypoxanthine in fish meat is described. The hypoxanthine sensor was prepared from xanthine oxidase immobilized by covalent binding to cellulose triacetate and a carbon paste electrode containing hydroxymethylferrocene. The xanthine oxidase membrane was retained behind a dialysis membrane at a carbon paste electrode. The sensor showed a current response to hypoxanthine due to the bioelectrocatalytic oxidation of hypoxanthine, in which hydroxymethyiferrocene served as an electron-transfer mediator. The limit of detection is 6 × 10^?7 M, the relative standard deviation is 2.8% (n=28) and the response is linear up to 7 × 10^?4 M. The sensor responded rapidly to a low hypoxanthine concentration (7 × 10^?4 M), the steady-state current response being achieved in less than 1 min, and was stable for more than 30 days at 5 ° C. Results for tuna samples showed good agreement with the value determined by the conventional method.     

Determination of kinetic parameters for pyrolysis of cellulose and cellulose treated with ammonium phosphate by differential thermal analysis and thermal gravimetric analysis

Activation energy E, pre-exponential factor k₀, and reaction order n for the pyrolysis of α-cellulose and cellulose modified with dihydrogen ammonium phosphate were determined by means of TGA and DTA. The results obtained are E = 53.5 kcal./mole, k₀ = 10^18.8 min.^?1, n = 1 for α-cellulose and E = 32 kcal./mole, k₀ = 10¹² min.^?1, n = 1 for modified cellulose. A new theory of DTA was also developed. This theory, in which it is concluded that the peak value of DTA curve coincides with the maximum rate of reaction, may be used not only for the present work but is generally applicable to DTA studies. Detailed procedures of experiment and theory are described.     

Seasonal fluctuation of DNA photodamage in marine plankton assemblages at Palmer Station, Antarctica

Ultraviolet radiation-induced DNA damage frequencies were measured in DNA dosimeters and natural plankton communities during the austral spring at Palmer Station, Antarctica, during the 1999-2000 field season. We found that the fluence of solar ultraviolet-B radiation (UV-B) at the earth's surface correlated with stratospheric ozone concentrations, with significant ozone depletion observed because of "ozone hole" conditions. To verify the interdependence of ozone depletion and DNA damage in natural microbial communities, seawater was collected daily or weekly from Arthur Harbor at Palmer Station, Antarctica, throughout "ozone season," exposed to ambient sunlight between 0600 and 1800 h and fractionated using membrane filtration to separate phytoplankton and bacterioplankton populations. DNA from these fractions was isolated and DNA damage measured using radioimmunoassay. Under low-ozone conditions cyclobutane dimer concentrations in bacterioplankton and phytoplankton communities were maximal. DNA damage measured in dosimeters correlated closely with ozone concentrations and UV-B fluence. Our studies offer further support to the theory that stratospheric deozonation is detrimental to marine planktonic organisms in the Southern Ocean.     

Synthesis of tricyclic compounds as steroid 5alpha-reductase inhibitors

A series of 4-phenoxybutyric acid derivatives attached to a tricyclic skeleton were prepared and evaluated as 5alpha-reductase inhibitors. Structure activity relationships for these compounds in terms of rat epididymis (type 2) 5alpha-reductase inhibitory activities reveal that 1) the substitution pattern at the 11-position of dibenz[b,e]oxepin influenced potency, 2) higher lipophilicity of the tricyclic skeleton improved potency, whereas the existence of a basic nitrogen atom in this skeleton was detrimental to potency, and 3) isobutyl substitution at the 8 positon of the azepine skeleton was tolerated. Among the tricyclic compounds studied, 4-[3-[5-benzyl-8-(2-methyl)propyl-10,11-dihydrodibenz[b,f]azepine- 2-carboxamido]phenoxy]butyric acid (26) was the most potent inhibitor of rat type 2 5alpha-reductase at 0.1 microM.     

Quantification of 2-hydroxyfluorene in human urine by column-switching high performance liquid chromatography with fluorescence detection

A high performance liquid chromatographic (HPLC) method for the quantification of 2-hydroxyfluorene (2-OHF) in normal human urine was established using deuterated 2-hydroxyfluorene (2-OHF-d9) as an internal standard with column-switching and fluorescence detection. The 2-OHF-d9 was synthesized by the metabolism of deuterated fluorene with cytochrome P450. The analytes were cleaned up on an ODS pre-column, via column-switching, and separated on an alkylamide-type reversed phase column. The internal standard eluted immediately prior to non-deuterated 2-OHF on the HPLC system and had nearly the same fluorescence characteristics as the non-deuterated 2-OHF. The detection limit was 0.03 nmol l(-1) (S/N = 3) and the calibration range of urine sample was from 0.2 to 50 nmol l(-1). The urine sample treatment involved enzymatic hydrolysis followed by solid phase extraction using a Sep-Pak C18 cartridge. 2-OHF was observed in the form of conjugates such as glucuronide and/or sulfate in human urine, and urinary metabolites were completely hydrolyzed for 2 h with beta-glucuronidase/aryl sulfatase. The proposed method was used to determine urinary 2-OHF in smokers and non-smokers, and showed that the urinary concentrations of 2-OHF in smokers were significantly higher than those in non-smokers (P < 0.01). Thus, the data suggest that urinary 2-OHF might be a sensitive and specific biological marker for the assessment of the exposure to polycyclic aromatic hydrocarbons.