A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane.

Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.

Structure of the alpha 1 subunit of horse Na,K-ATPase gene.

Genomic DNA for Na,K-ATPase alpha 1 subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the alpha 1 subunit of rat Na,K-ATPase as probe. The gene spans 30 kb and consists of 23 exons and 22 intervening sequences. Intron-exon boundaries were analyzed. The protein-coding nucleotide sequence encodes 1016 amino acids with an Mr of 112,264. The putative amino acid sequence of horse alpha 1 is 96-97% homologous to those of other mammalian species.

CPP32 activation during dolichyl phosphate-induced apoptosis in U937 leukemia cells.

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre-CPP32 to generate p17. Peptide inhibitors YVAD-cmk and Z-Asp-CH2-DCB (specific to ICE) and DEVD-CHO (specific to CPP32) blocked the dolichyl phosphate-induced apoptosis. The dolichyl phosphate-induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2',5'-dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.

DNA cleavage and 8-hydroxydeoxyguanosine formation caused by tamoxifen derivatives in vitro.

DNA damage caused by tamoxifen and its derivatives was examined by estimating the conversion of supercoiled pUC18 plasmid DNA to linear form by means of agarose gel electrophoresis. N-Desmethyltamoxifen induced DNA cleavage and its effect was enhanced by the addition of reducing agents such as dithiothreitol, NADPH and 2-mercaptoethanol. 4-Hydroxytamoxifen itself had little effect, but the cleavage was slightly enhanced by the addition of reducing agents. DNA damage was higher with alpha-hydroxytoremifene than with alpha-hydroxytamoxifen, which had a prominent effect only at high concentration. The cleavage by alpha-hydroxy derivatives were not enhanced by reducing agents. No damage was induced by tamoxifen, toremifene, 3-hydroxytamoxifen or N-desmethyltoremifene. The DNA cleavage by N-desmethyltamoxifen was inhibited by the addition of EDTA, mannitol, sodium azide, methionine, catalase and superoxide dismutase. The formation of 8-hydroxy-2'-deoxyguanosine was also examined with calf thymus DNA in vitro. A slight increase of its level was found with 4-hydroxytamoxifen in the presence of dithiothreitol and also with N-desmethyltamoxifen in the presence of NADPH, but alpha-hydroxytoremifene and alpha-hydroxytamoxifen were ineffective. These experimental data suggest that among metabolites of tamoxifen, N-desmethyltamoxifen and probably also 4-hydroxytamoxifen cause oxidative DNA damage in which redox cycling is involved. The DNA damage by alpha-hydroxytoremifene appears to involve a different mechanism from that by N-desmethyltamoxifen. Tamoxifen and toremifene are possibly metabolized to the forms contributing to DNA damage.

Interaction of beta-eudesmol with Na+,K(+)-ATPase: inhibition of K(+)-pNPPase activity.

The effect of beta-eudesmol, one of the major components in So-jutsu (Atractylodis Lanceae Rhizoma), on K(+)-dependent p-nitrophenyl phosphatase (K(+)-pNPPase) activity was studied. It inhibited K(+)-pNPPase activity with an I50 value of 4.1 x 10(-4) M. The inhibition rate decreased as the K+ concentration was increased, whereas greater inhibition was observed with high concentrations of either Na+ or ATP. The Ki values for Na+ in the presence of 0, 0.1 and 1 mM ATP were 140, 260 and 310 mM, respectively, but with the addition of beta-eudesmol, these values decreased to 90 mM regardless of the ATP concentration. This study on K(+)-pNPPase activity supports the conclusion obtained from the study on Na+,K(+)-ATPase activity (Satoh K et al., Biochem Pharmacol 44: 373-378, 1992) that is, beta-eudesmol interacts with enzyme in the Na.E1 form and inhibits the reaction step Na.E1----Na.E1-P. Furthermore, in the study of the effects of K+ and beta-eudesmol on K(+)-pNPPase activity, it was confirmed that beta-eudesmol prevents the conformational change of Na.E1----K.E2.

Inhibition of H+,K+ -ATPase by hinesol, a major component of So-jutsu, by interaction with enzyme in the E1 state.

Hinesol, a major component of the crude drug "So-jutsu" (Atractylodis Lanceae Rhizoma), strongly inhibited H+,K+-ATPase activity with a IC50 value of 5.8x10(-5) M. It also inhibited Na+,K+-ATPase, Mg2+-ATPase, Ca2+-ATPase, and H+-ATPase activities, although the inhibition rate was lower. No effects on alkaline or acid phosphatase activities were observed. The mechanism by which hinesol inhibited H+,K+-ATPase activity was studied in detail. The inhibition was uncompetitive with respect to ATP, and it increased as the Mg2+ concentration was raised, whereas it was not affected by the K+ concentration. The activity of K+-dependent p-nitrophenyl phosphatase (K+-pNPPase), a partial reaction of H+,K+-ATPase, was inhibited by hinesol noncompetitively with respect to pNPP (IC50 value of 1.6x10(-4) M), and competitively with respect to K+, whereas it was not affected by the Mg2+ concentration. These results suggest that hinesol is a relatively specific inhibitor of H+,K+-ATPase. It appears that hinesol reacts with enzyme in the E1 state in the presence of ATP and Mg2+ and forms the complex hinesol-H+ E1-ATP or hinesol x E1-P, blocking the conformational change to the E2 state. Furthermore, hinesol enhanced the inhibitory effect of omeprazole on H+,K+-ATPase, and the inhibitory site of hinesol was different from that of omeprazole. The effect of So-jutsu as an anti-gastric ulcer agent may be ascribed to the inhibitory effect of hinesol on H+,K+-ATPase activity.

Specific inhibition of Na+,K(+)-ATPase activity by atractylon, a major component of byaku-jutsu, by interaction with enzyme in the E2 state.

Atractylon, a major component of the crude drug "Byaku-jutsu" (rhizomes of Atractylodes japonica), strongly inhibited Na+,K(+)-ATPase activity with an I50 value of 8.9 x 10(-6) M. It also inhibited Mg(2+)-ATPase, H+,K(+)-ATPase, H(+)-ATPase and Ca(2+)-ATPase activities, but less potently. No effects on alkaline and acid phosphatase activities were observed. The inhibition of Na+,K(+)-ATPase activity by atractylon was noncompetitive with respect to ATP and was greater with increasing K+ concentration, whereas it was not affected by Na+ concentration. The activity of K(+)-dependent p-nitrophenyl phosphatase, a partial reaction of Na+,K(+)-ATPase, was inhibited noncompetitively with respect to substrate (I50 value of 1.8 x 10(-5) M), and the inhibition rate was independent of the K+ concentration. Furthermore, atractylon increased the Ki value for Na+ from 130 to 190 mM, but did not alter the Ki value for ATP. Inhibition of the phosphoenzyme formation by atractylon was greater at 0.1 M than at 1 M NaCl. K(+)-dependent dephosphorylation (E2-P to K.E2) was inhibited by atractylon, whereas ADP-sensitive (Na.E1-P to Na.E1) and non-specific dephosphorylation steps were not affected. These results suggest that atractylon, a specific inhibitor of Na+,K(+)-ATPase, interacts with enzyme in the E2 state and inhibits the reaction step from E2-P to K.E2.

Structure-activity relationships of nitrosamines and nitramines which stimulate UDP-glucuronosyltransferase activities in vitro.

Examination of twelve nitrosamines and seven nitramines revealed that nitramines modify UDP-glucuronosyltransferase activity in a manner similar to that of nitrosamines. Only N,N-diethyl-substituted nitrosamine and nitramine significantly stimulated transferase activity toward 2-aminophenol and 4-nitrophenol but not toward phenolphthalein and androsterone. Elongation of the alkyl chains or introduction of carboxy, hydroxy, or oxo groups into the alkyl chains did not result in stimulatory ability, and some of these compounds inhibited the transferase activity.

Inhibition of Na+,K(+)-ATPase activity by beta-eudesmol, a major component of atractylodis lanceae rhizoma, due to the interaction with enzyme in the Na.E1 state.

beta-Eudesmol, a major component of the crude drug "So-jutsu" (Atractylodis Lanceae Rhizoma), inhibited Na+, K(+)-ATPase activity most strongly among the various kinds of phosphatases examined. It also inhibited Ca(2+)-ATPase and H+, K(+)-ATPase, but to a lesser extent. Its effect on Mg(2+)-ATPase was minute. No effects on H(+)-ATPase or alkaline and acid phosphatase activities were observed. The effects of beta-eudesmol on horse kidney Na+, K(+)-ATPase were studied in detail, and the following results were obtained: (1) beta-eudesmol inhibited the Na+, K(+)-ATPase activity with an I50 value of 1.6 x 10(-4) M. The mode of its inhibition was uncompetitive with respect to ATP; (2) it prevented the stimulation of enzyme activity by Na+. The inhibition gradually increased in accord with the increase of Na+ concentration, and it was constant when Na+ was higher than 6.3 mM; (3) it did not alter the K+ concentration necessary for half-maximal activation (K0.5 for K+); and (4) it inhibited the enzyme activity with a mode of action different from ouabain. Phosphorylation of enzyme with [gamma-32P]ATP was inhibited by beta-eudesmol with an I50 of 1.4 x 10(-4) M. The inhibition was greater in 1 M NaCl than in 0.1 M NaCl. It had no effects on dephosphorylation steps, i.e. none of the non-specific, the ADP-sensitive (Na.E1-P----Na.E1) and the K(+)-dependent (E2-P----K.E2) dephosphorylation processes were affected. These results suggest that beta-eudesmol, a relatively specific inhibitor of Na+, K(+)-ATPase, interacts with the enzyme in the Na.E1 form and inhibits the reaction step Na.E1----Na.E1-P.

Inhibition of Na+,K(+)-ATPase by 1,2,3,4,6-penta-O-galloyl-beta-D-glucose, a major constituent of both moutan cortex and Paeoniae radix.

The inhibition of Na+,K(+)-ATPase activity by various constituents of Moutan Cortex and Paeoniae Radix was studied. 1,2,3,4,6-Penta-O-galloyl-beta-D-glucose (PGG), a major component of both crude drugs, strongly inhibited Na+,K(+)-ATPase activity (IC50 = 2.5 x 10(-6) M), whereas galloylpaeoniflorin, benzoic acid, and catechin were weakly inhibitory, and albiflorin, oxypaeoniflorin, paeoniflorin, paconol, and phenol were ineffective. The inhibition of Na+,K(+)-ATPase activity by PGG was decreased in the presence of BSA or phospholipids. The inhibition mode of PGG was noncompetitive with respect to ATP. The K0.5 value for Na+ was increased by the addition of PGG from 9.1 to 12.3 mM, whereas that for K+ was not altered. PGG also inhibited K(+)-dependent p-nitrophenyl phosphatase activity with an IC50 value of 5.3 x 10(-6) M, and the extent of the inhibition increased at higher concentrations of K+. The K0.5 value for K+ was decreased by the addition of PGG from 3.3 to 2.0 mM. These results suggested that the inhibition of Na+,K(+)-ATPase activity is caused by interaction of PGG with the enzyme in the E2 state. The inhibitory effect of Moutan Cortex or Paeoniae Radix is considered to be mainly attributable to PGG.

Inhibition of calmodulin stimulation of phosphodiesterase and Ca2+, Mg2+-ATPase activities and shape change of erythrocyte ghosts by chloroquine.

The effects of chloroquine on calmodulin (CaM)-related enzyme activities and the shape of human erythrocytes have been studied. It was found that the CaM activation of rat brain phosphodiesterase was abolished by the addition of chloroquine. CaM was included in the assay of phosphodiesterase activity at the concentration that gave half-maximal activation. The concentration of chloroquine that caused 50% inhibition of CaM stimulation of phosphodiesterase was 7 X 10(-5)M. The type of inhibition was competitive with respect to CaM. The CaM-stimulated Ca2+, Mg2+-ATPase in erythrocyte membrane was also inhibited by chloroquine, the 50% inhibitory concentration of which was about 2 X 10(-4)M. Its mode of action was also competitive with respect to CaM. The shapes of erythrocyte ghosts prepared by hypotonic hemolysis were examined in a solution consisting of 2 mM MgCl2, 154 mM NaCl and 10 mM Tris-HCl (pH 7.4); they were discocytic in the presence of 2 mM ATP and in its absence. They were converted to the invaginated form by the addition of chloroquine in the concentration range of 1 X 10(-4)-5 X 10(-4)M. This concentration is similar to that which caused the inhibition of CaM activation of Ca2+, Mg2+-ATPase.

Certain styrene oligomers have proliferative activity on MCF-7 human breast tumor cells and binding affinity for human estrogen receptor.

To examine the estrogenic activities of styrene oligomers, we carried out cell proliferation assays with estrogen-sensitive MCF-7 cells and competitive binding assays to human estrogen receptor [alpha] (hER[alpha]). The styrene oligomers tested were 1,3-diphenyl propane (SD-1), 2,4-diphenyl-1-butene (SD-2), cis-1,2-diphenyl cyclobutane (SD-3), trans-1,2-diphenyl cyclobutane (SD-4), 2,4,6-triphenyl-1-hexene (ST-1), 1a-phenyl-4a-(1'-phenylethyl)tetralin (ST-2), 1a-phenyl-4e-(1'-phenylethyl)tetralin (ST-3), 1e-phenyl-4a-(1'-phenylethyl)tetralin (ST-4), 1e-phenyl-4e-(1'-phenylethyl)tetralin (ST-5), 1e,3e,5a-triphenylcyclohexane (ST-6), and 1e,3e,5e-triphenylcyclohexane (ST-7). In the MCF-7 cell proliferation assay, styrene trimers (ST-1, ST-3, ST-4, and ST-5) had the highest proliferative activities of the compounds tested. The relative potency of these chemicals was 0.0002-0.0015%, which was comparable with that of bisphenol A (0.0001-0.0025%), and their relative proliferative effect was 51-104%. Styrene dimers (SD-3 and SD-4) also significantly increased the cell yields. However, SD-1, SD-2, ST-2, ST-6, and ST-7 had insignificant proliferative activities. The competitive binding assay revealed the binding affinity of some styrene oligomers for hER[alpha]. The order of their binding potency for hER[alpha] was as follows: ST-4 > ST-2 > ST-3 > ST-5 > ST-1 > SD-3 > SD-4 > SD-2 > SD-1. ST-6 and ST-7 did not appear to bind to hER[alpha]. The present studies indicate that styrene dimers SD-3 and SD-4 and styrene trimers ST-1, ST-3, ST-4, and ST-5 have estrogenic activity on MCF-7 cells and binding affinity for hER[alpha]. These compounds might be endocrine disrupters.

Immobilization of antibody to a plastic surface by toluene 2,4-diisocyanate and its application to radioimmunoassay.

A novel procedure to fix antibody on a plastic surface was developed, using a polystyrene tube treated with 0.5% toluene 2,4-diisocyanate (TC) dissolved in carbon tetrachloride. Using 125I-labelled IgG, we found that the rate of immobilization of IgG was significantly higher than that obtained by simple adsorption or coupling by glutaraldehyde in the presence of high concentrations of IgG. The polystyrene tube coated with anti-rabbit gamma-globulin goat serum allowed a simple separation of the tracer bound to the first antibody from the free tracer in a radioimmunoassay of thyroid-stimulating hormone and prolactin, when the coating was done by the TC method but not by the simple adsorption or glutaraldehyde methods.

DNA cleavage by phenylhydroquinone: the major metabolite of a fungicide o-phenylphenol.

The interaction of o-phenylphenol (OPP) and its metabolites with DNA was examined. As a model system, the reactivities of OPP and its metabolites with DNA were studied by using pUC18 DNA. The major metabolite formed in vitro from OPP by mixed function oxidase was phenylhydroquinone (PHQ). This result corresponds to the findings that PHQ in the form of glucuronide conjugate was the main product detected in bladder of OPP fed rats in vivo. When supercoiled pUC18 DNA (form I) was incubated with PHQ at concentrations from 1 X 10(-6) M to 2 X 10(-1) M, DNA strand scission by PHQ was observed at a concentration as low as 1 X 10(-5) M and the amount of linear form (form III) increased with increasing PHQ concentration. PHQ causes DNA strand scission. The DNA cleavage by OPP and phenyl-p-benzoquinone (PBQ) was barely detectable. The DNA cleavage by PHQ was inhibited by superoxide dismutase (SOD), catalase and several oxygen radical scavengers such as polyethylene glycol, tert-butanol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine. The production of superoxide radical from PHQ was confirmed by cytochrome c reduction assay. These results indicate that the oxygen radicals such as superoxide, hydroxyl radicals and some others generated in the process of oxidation of PHQ in aqueous solution are responsible for the DNA cleavage. In order to identify the sites of cleavage of DNA by PHQ, a 5'-end 32P-labeled 206 base-pair EcoRI-BglI fragment of pUC18 DNA was incubated with PHQ. The DNA was then analyzed by sequencing gel electrophoresis followed by autoradiography. When the DNA was incubated with PHQ and further treated with piperidine, cleavage was detected relatively more frequently at guanine residues. The attack seemed to occur at guanine residues in general, but was not restricted to guanines with specific residues in the vicinity.

On the nature of rat hepatic and mouse olfactory sulfotransferases.

Rat hydroxysteroid sulfotransferase (HS-SULT) cDNAs, ST-40 and ST-20 are 90% identical in amino acid sequences and show different substrate specificities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). ST-40 enzyme is active toward the three substrates, whereas ST-20 enzyme is preferentially active for CS. First we prepared mutants of well conserved histidine, lysine and asparagine by site-directed mutagenesis. Secondly we constructed 20 chimeric HS-SULTs by reciprocal exchange of five protein domains between ST-20 and ST-40 enzymes. The studies on the expressed mutant and chimeric enzymes indicate the importance of the C-terminal region for the substrate specificity and the involvement of multiple regions for the enzyme activities. Next we determined the genetic loci of ST-40 and ST-20 by fluorescence in situ hybridization. Biotinylated ST-20 and ST-40 probes gave a pair of fluorescent spots on the same region of rat chromosome 1 and the loci of these genes were localized to the same chromosomal region of 1q21.3 --> q22.1. Finally we studied mouse olfactory phenol SULT (P-SULT). It was immunolocalized in the cytoplasm of mouse olfactory sustentacular cells and mouse nasal cytosols show high SULT activities toward phenolic aromatic odorants. We subsequently isolated a mouse P-SULT cDNA from mouse olfactory cDNA library. It encodes 304 amino acid polypeptide and is 94% identical with rat ST1C1 in amino acid sequences.

Contribution of oxygen radicals to DNA cleavage by quinone compounds derived from phenolic antioxidants, tert-butylhydroquinone and 2,5-di-tert-butylhydroquinone.

The effects of synthetic phenolic antioxidants, tert-butylhydroquinone (TBHQ), 2,5-di-tert-butylhydroquinone (DTBHQ) and 3-tert-butyl-4-hydroxyanisole (BHA), on DNA cleavage were examined with supercoiled plasmid DNA, pUC18, in vitro. Extensive single and double strand breaks of DNA by TBHQ were observed and almost all the DNA was converted to the linear form at 10(-2) M. The cleavage was stimulated by both CuCl2 and FeCl2, though the effect of FeCl2 was smaller. Metal ion chelators and some oxygen radical scavengers inhibited the cleavage. The generation of TBHQ semiquinone radical and hydroxyl radical in the presence of copper was demonstrated by ESR spectroscopy. DTBHQ also caused DNA cleavage, though its effect was much smaller than that of TBHQ. BHA had no effect in the experimental systems employed. Oxygen radicals were considered to contribute to the DNA cleavage by TBHQ and DTBHQ.

Formation of 8-hydroxydeoxyguanosine in calf thymus DNA treated with tert-butylhydroquinone, a major metabolite of butylated hydroxyanisole.

Oxidative DNA damage caused by butylated hydroxyanisole (BHA), 2-tert-butyl(1,4)hydroquinone (TBHQ, a metabolite of BHA) and 2,5-di-tert-butyl(1,4)hydroquinone (DTBHQ), as well as 2,6-di-tert-butyl(1,4)benzoquinone (BHTQ, a metabolite of butylated hydroxytoluene), was evaluated by measuring the formation of 8-hydroxydeoxyguanosine (8OHdG) in calf thymus DNA. 8OHdG formation was greatly increased by TBHQ in a concentration-dependent manner. This effect was strongly enhanced by CuCl2 and suppressed by EDTA, bathocuproinedisulfonic acid disodium salt, methionine, glutathione reduced form or catalase, but was not affected by mannitol, sodium benzoate or sodium azide. Thus, TBHQ-induced 8OHdG formation may be mediated by copper. DTBHQ also induced the formation of 8OHdG, though to a much lesser extent than TBHQ, and its effect was stimulated by CuCl2. BHA had a small enhancing effect at high concentration, only in the presence of CuCl2, whereas in the case of BHTQ, it occurred both in the presence of CuCl2 and FeCl2.

Changes in enzyme activities in the urine and tissues of rats fed sodium o-phenylphenate.

The activities of certain enzymes in the urine and tissues of rats given 2% sodium o- phenylphenate in the diet for 20 wk were examined. Urinary gamma-glutamyl transpeptidase (gamma GTP) decreased immediately after the start of feeding of the treated diet and its activity remained low for 20 wk. The gamma GTP and alkaline phosphatase (ALP) activities in kidney homogenate decreased to about 80% of the control at 20 wk, but G6PD activity was significantly increased; Na,K-ATPase was unchanged. On the other hand, the gamma GTP activity in the liver homogenate of treated rats was increased to about eight times that of the controls, the G6PD activity showed a significant increase, but the ALP and Na, K-ATPase activities were not significantly different from the control values. The glutathione concentration in the livers of treated rats was significantly reduced.

Study on anti-androgenic effects of bisphenol a diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives using cells stably transfected with human androgen receptor, AR-EcoScreen.

We studied in vitro hormonal activity of bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE), which are used as a material of interior coating for food cans. We also examined related compounds such as 2,2-bis[4-(3-chloro-2-hydroxypropoxy)phenyl]propane (BADGE.2HCl), and bis[4-(3-chloro-2-hydroxypropoxy)phenyl]methane (BFDGE.2HCl) etc. For this purpose, we constructed two stably transfected CHO-K1 cell lines (AR-EcoScreen for androgenic activity and c-luc for cell toxicity evaluation). One stably expresses luciferase with induction of androgen. The other stably expresses luciferase without androgen induction. Also, we have determined the androgenic and anti-androgenic effects of the test chemicals by reporter gene assay with these cell lines. None of the chemicals tested by this assay exhibited androgen agonistic activity. However, BADGE.2HCl and BFDGE.2HCl had the conspicuous antagonistic activity for androgen. These compounds had a high binding affinity for androgen receptor. Furthermore, these two compounds did not show the estrogenic activity in vitro assays. On the contrary, bisphenol A and bisphenol F exhibited anti-androgenic activity in vitro in addition to the estrogenic activity. These results suggest that these chlorohydroxy compounds of BADGE and BFDGE act as androgen antagonist through the process of binding to androgen receptor.

Inhibition of aromatase activity by green tea extract catechins and their endocrinological effects of oral administration in rats.

We orally administered polyphenone-60 (P-60), green tea extract catechins, in the diet (0, 1.25 and 5%) to male rats for 2, 4 and 8 weeks initiated at 5 weeks old. It was found that a 5% dose to male rats for 2-8 weeks induced goiters and decreased weights of the body, testis and prostate gland. Endocrinologically, elevating plasma thyroid stimulating hormone (TSH), luteinizing hormone (LH) and testosterone levels and decreasing tri-iodothyronine (T(3)) and thyroxine (T(4)) levels were induced by this treatment. We also found that P-60 as a whole and some of its constituents exhibited inhibitory effects on human placental aromatase activity by in vitro assay. The concentration of P-60 that required producing 50% inhibition of the aromatase activity (IC(50) value) was 28 microg/ml. The IC(50) values of (-)-catechin gallate (Cg), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCg) and (-)-gallocatechin gallate (GCg) were 5.5 x 10(-6), 1.0 x 10(-4), 6.0 x 10(-5) and 1.5 x 10(-5) M, respectively. (-)- Epicatechin gallate (ECg) at 1.0 x 10(-4) M produced 20% inhibition. (-)-Epicatechin (EC) and (+)-catechin (CT) exhibited no effects on aromatase activity. The endocrinological changes observed in vivo were in conformity with antithyroid effects and aromatase inhibition effects of P-60 and its constituents.