Responses of estrogen sensitive tissues in female Wistar rats to pre- and postnatal isoflavone exposure.

Effects of isoflavones on estrogen sensitive tissues are discussed controversially. This study was designed to investigate tissue specific effects of an isoflavone exposure through different periods of life in female Wistar rats and to compare the effects of genistein (GEN) to those of mixed dietary isoflavones, GEN and daidzein (DAI). One group received an isoflavone-free diet (IDD), another was fed an isoflavone-rich diet (IRD) and the third group an IDD supplemented with GEN (GEN(d)) prior to mating, throughout pregnancy and up to weaning. The offspring were kept on the respective diets during growth, puberty and adulthood. The weight of the uterus, the height of the uterine and vaginal epithelium, the bone mineral density of the tibia, and the expression of the estrogen sensitive gene CaBP9K in the liver were determined. At d21, the uterine weight, the uterine epithelium and the expression of CaBP9K in the liver were significantly stimulated in GEN(d) animals compared to IDD and IRD. Interestingly, bone mineral density was increased in GEN(d) and in IRD animals. Around puberty (d50) neither uterine wet weights nor trabecular bone density differed significantly among the isoflavone groups and the IDD control. At d80 no significant differences in uterine weight were observed among IDD, GEN(d) and IRD animals. However, bone mineral density was increased in GEN(d) and IRD animals. In summary, our results demonstrate that lifelong dietary exposure to isoflavones can affect estrogen sensitive tissues, apparently in a tissue selective manner. With respect to health risk and benefit our data indicate that an increased bone mineral density can be achieved by lifelong exposure to an IRD, which, in contrast to GEN supplementation, does not seem to stimulate the proliferation of the uterine epithelium.

Antioxidant activities of major thyme ingredients and lack of (oxidative) DNA damage in V79 Chinese hamster lung fibroblast cells at low levels of carvacrol and thymol.

The leafy parts of thyme and its essential oil have been used in foods for the flavour, aroma and preservation and also in folk medicines. In the present study the genotoxicity of thymol and carvacrol was examined using comet assay. In V79 Chinese hamster lung fibroblast cells treated with 1, 5, 25 microM thymol and carvacrol, only 25 microM thymol caused some clastogenic DNA damage. For detection of oxidative DNA damage, the comet assay with formamido pyrimidine glycosylase (Fpg) protein was used: When V79 cells were treated with 1, 5, 25 microM thymol and carvacrol and post-treated with Fpg enzyme, no significant increase of Fpg-sensitive sites was observed at all concentrations studied. Reactive oxygen species (ROS) generation decreased slightly in the presence of thymol (1-100 microM) and carvacrol (5 microM) between 1 and 4h, yet increased at the highest 100 microM concentration of carvacrol after 24h. Thymol and carvacrol displayed a concentration dependent antioxidant capacity, whilst gamma-terpinene which lacks a phenolic group did not show any antioxidant capacity in the trolox equivalent antioxidant capacity (TEAC) assay. The results of this study indicate a lack of clastogenic activity for thymol and carvacrol at biologically relevant concentrations, and a moderate antioxidant activity in vitro.

Interaction of phytoestrogens and other environmental estrogens with prostaglandin synthase in vitro.

The phytoestrogens daidzein, genistein, equol and coumestrol were found to stimulate microsomal prostaglandin H synthase (PHS) in vitro in a concentration-dependent manner when PHS-activity was measured by arachidonic acid-dependent oxygen uptake. These compounds were co-oxidized by PHS and the conversion of parent compounds was measured by HPLC analysis. The stimulation of PHS-cyclooxygenase by these compounds was partially reversed at high concentrations probably due to their antioxidant properties causing inhibition. In contrast, the monomethyl ethers of daidzein and genistein, formononetin and biochanin A, had little or weakly inhibitory effect on PHS, and appear to be no or poor co-substrates for PHS. Compared to the equine estrogen equilin, its metabolite d-equilenin was poorly metabolized by PHS and inhibited rather than stimulated PHS-cyclooxygenase activity in vitro. The resorcylic acid lactones zearalenone and zeranol, on the other hand, were surprisingly good inhibitors of PHS-cyclooxygenase. Furthermore, zeranol inhibited both the arachidonic acid and the hydrogen-peroxide-dependent oxidation of DES in contrast to indomethacin which inhibited only cyclooxygenase-dependent co-oxidation of DES. The results of this in vitro study are discussed in the context of data on synthetic and steroidal estrogens and support the idea that PHS-activity may be modulated by interaction with certain estrogenic compounds.

Multiple pathways for the oxidative metabolism of estrogens in Syrian hamster and rabbit kidney.

Microsomal preparations from hamster kidney, a target tissue for the carcinogenic action of stilbene-type and steroidal estrogens, catalyze the oxidative metabolism of diethylstilbestrol (DES). The formation of the major metabolite Z,Z-dienestrol and of reactive intermediates capable of protein binding were mediated by enzyme activities requiring nicotinamide-adenine dinucleotide phosphate (reduced form-NADPH), cumene hydroperoxide, or arachidonic acid (ARA). In addition, hydroxylated DES metabolites were detected in NADPH-supplemented incubations. The NADPH-dependent oxidation of DES was inhibited by SKF 525A and metyrapone. Monooxygenase-catalyzed metabolism was apparently responsible for the majority of DES oxidation in microsomes from whole hamster kidneys in vitro and this activity is preferentially localized in the kidney cortex. However, ARA-dependent, i.e., prostaglandin H synthase (PHS) mediated oxidation of DES and of the catechol estrogen 2-hydroxyestrone was demonstrated as well in the medulla of both rabbit and hamster kidney. It is proposed that monooxygenase and PHS activities act in concert in the metabolic activation of carcinogenic estrogens. This appears to apply in particular to steroidal estrogens, since catechol estrogens formed by monooxygenases are further oxidized to reactive intermediates by PHS and other peroxidatic enzymes.

Prostaglandin H synthase catalyzes regiospecific release of tritium from labeled estradiol.

Prostaglandin H synthase (PHS) from ram seminal vesicle microsomes was found to catalyze the release of tritium (3H) from estradiol (E2) regiospecifically labeled in position C-2 or C-4 of ring A but not from positions C-17 alpha, C-16 alpha, or C-6,7. Formation of 3H2O from ring A of E2 is dependent upon native enzyme supplemented with either arachidonic acid, eicosapentaenoic acid, or hydrogen peroxide and proceeds very rapidly as do other cooxidation reactions catalyzed by PHS-peroxidase. The 3H-loss from ring A of E2 reflecting oxidative displacement of this isotope by PHS increases linearly up to 100 microM under our conditions (8-45 nmol/mg x 5 min). Loss of tritium in various blanks is negligible by comparison. Indomethacin (0.07 and 0.2 mM) inhibited the PHS-dependent release of 3H2O from estradiol but less efficiently than it inhibited DES-cooxidation measured in parallel incubations under similar conditions. Addition of EDTA (0.5 mM) had no effect on the regiospecific transfer of 3H from E2 or on DES-oxidation; ascorbic acid (0.5 mM) or NADH (0.33 mM) clearly inhibited both reactions and to a similar extent. These data suggest that estradiol-2/4-hydroxylation can be catalyzed by PHS in vitro probably via its peroxidase activity and point to PHS as an enzyme that could contribute to catechol estrogen formation in vitro by tissue preparations in the presence of unsaturated fatty acids or peroxides.

Prostaglandin-H-synthase competent cells derived from ram seminal vesicles: a tool for studying cooxidation of xenobiotics.

Prostaglandin-H-synthase (PHS) peroxidase has been suggested to mediate drug metabolism particularly in extrahepatic tissues low in monooxygenase (MFO) activity. PHS can oxidize various xenobiotics in vitro; its contribution in vivo is still uncertain and is currently assessed by differences in the MFO- and PHS-catalyzed product/adduct formation of a few suitable substrates. Cells in culture that are PHS competent but MFO deficient can provide an additional approach for further investigating the role of PHS in the metabolic activation of foreign compounds. To this end, a cell line has been derived from ram seminal vesicles (SEMV), a tissue known as a good source of PHS but shown to be devoid of MFO activity. SEMV cells can be cultured in IBR or in RPMI medium supplemented with fetal calf serum, and have been subcultured until passage 30. The arachidonic acid (AA) metabolism in these cells has been characterized: besides incorporation in the lipid pool, AA was mainly metabolized to prostaglandin (PG) E2; minor products were PGF2 alpha and the lipoxygenase products 12- and 15-HETE. The PGE2 production (17 nmol/10(6) cells.24 h) of SEMV cells (passage 10) exceeded at least 10-fold that of other cells cultured under similar conditions. These data, indicative of high PHS activity, suggest that the cells can be a useful tool for future studies on the objectives outlined above.

Oxidative metabolism of diethylstilbestrol by prostaglandin synthetase.

The cooxidative metabolism of the transplacental carcinogen, diethylstilbestrol (DES), was examined using ram seminal vesicle microsomes. The major extractable metabolite was beta-dienestrol (Z,Z-DIES) and represented about 35% of the added DES in 3-min incubations supplemented with arachidonic acid. Its formation was dependent upon the presence of arachidonic acid, whereas reduced nicotinamide adenine dinucleotide phosphate failed to elicit Z,Z-DIES above background. Indomethacin and 1-phenyl-3-pyrazolidone, known inhibitors of prostaglandin synthetase, blocked Z,Z-DIES formation, probably by inhibiting the cyclooxygenase and the hydroperoxidase activities, respectively. Hydrogen peroxide and 15-hydroperoxyarachidonic acid (cosubstrates of the prostaglandin synthetase-hydroperoxidase), when replacing arachidonic acid in incubations, also supported oxidative metabolism of DES catalyzed by ram seminal vesicle microsomes. 1-Phenyl-3-pyrazolidone, but not indomethacin, inhibited the 15-hydroxyperoxyarachidonic acid-dependent formation of Z,Z-DIES. Incubation conditions which supported efficient Z,Z-DIES formation also resulted in the formation of 3,3-di(p-hydroxyphenyl)hexan-4-one and the cis-isomer of DES as well as nonextractable, protein-associated radioactivity indicating the presence of reactive intermediates. The implications of the peroxidative metabolism of DES for its toxic activity are obvious.