Testicular toxicity and mutagenicity of steroidal and non-steroidal estrogens in the male mouse.

The mutagenicity and toxicity of diethylstilbestrol (DES), 17 beta-estradiol and zeranol on the male mouse germ cells were investigated with meiotic micronucleus assays in vivo and in vitro, sperm-head abnormality test and morphometry. Further, the developmental effects of DES on testicular morphology were explored. Micronucleus induction was observed at 10(-7) M concentration of DES and 17 beta-estradiol in vitro, but other treatments yielded negative results. The micronucleus assay in vivo revealed a small number of micronuclei in early haploid spermatids 17 days after a single subcutaneous injection of DES 50 mg/kg, whereas estradiol and zeranol gave negative results. The sperm-head abnormality rates were significantly elevated 5 weeks after treatments with high doses of DES, 17 beta-estradiol and zeranol, and testicular morphometry revealed transient changes in the volume densities of testicular tissue components. Prenatal and neonatal estrogen administration resulted in permanent alterations in seminiferous epithelium and dilatation of the rete testis, but did not affect micronucleus or sperm-head abnormality rates. The mutagenicity and toxicity of hormones in the mouse testis paralleled the hormonal activity of these compounds. Early estrogenization was the most sensitive toxicity test, followed by in vitro meiotic micronucleus induction, whereas the sperm-head abnormality assay and morphological analysis did not reveal subtle changes.     

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.     

Inhibition of carbonic anhydrase II by steroidal and non-steroidal sulphamates

Carbonic anhydrases (CAs) are expressed by many solid tumours where they may act to confer a growth advantage on malignant tissues. In this study we have examined the ability of a series of steroidal and non-steroidal sulphamates (originally developed as steroid sulphatase inhibitors) and related compounds to inhibit human CAII (hCAII) activity in vitro. Using a 96-well plate assay, oestrone-3-O-sulphamate (EMATE) and two coumarin-based sulphamate drugs (667 COUMATE and STX 118) were found to have IC(50) values of 25-59 nM for the inhibition of hCAII activity. These compounds therefore have a similar CAII inhibitory potency to that of acetazolamide (IC(50)=25 nM), a known hCAII inhibitor. Docking studies have been performed with selected compounds to the crystal structure of hCAII and excellent correlation of scores with biological activity was observed. This agrees with our recent observations when we were the first to report the inhibition of hCAII by STS inhibitors. These studies and initial results with docking to the crystal structure of the extracellular domain of hCAXII indicate that the STS sulphamate ester inhibitors should also be interesting candidates to pursue as inhibitors of CA isozymes that are over-expressed in human tumours.     

Active nonaromatic intermediates in the conversion of steroidal estrogens into catechol estrogens

A mechanism is proposed for mixed-function oxidase-catalyzed formation of the catechol estrogens 2-hydroxy- and 4-hydroxyestradiol from estradiol. This mechanism involves nonaromatic epoxyenones as intermediates. The isomeric 1 alpha,2 alpha-epoxy-17 beta-hydroxyestr-4-en-3-one and 1 beta,2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (the latter as its 17-acetate) were synthesized from 17 beta-hydroxy-5 alpha-estran-3-one. The isomeric 4 alpha,5 alpha-epoxy-17 beta-hydroxyestr-1-en-3-one and 4 beta,5 beta-epoxy-17 beta-hydroxyestr-1-en-3-one were prepared from 19-nortestosterone. From incubations of [6,7-3H]estradiol with microsomes from MCF-7 human breast cancer cells, which principally catalyze the formation of 2-hydroxyestradiol from estradiol, we were able to isolate a 3H-labeled product with the chromatographic properties of 1 beta, 2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (as its 17-acetate). The soluble protein fraction of homogenates of rat liver, which is devoid of estrogen 2-/4-hydroxylase activity, has been shown to catalyze the formation of 2- and 4-hydroxyestradiol from the 1 alpha,2 alpha-epoxide and from the 4 alpha,5 alpha- and 4 beta,5 beta-epoxides, respectively. We suggest that these results taken together strongly support a role for epoxyenones as intermediates in the formation of catechol estrogens.     

Mechanical stimulation of skeletal muscle cells mitigates glucocorticoid-induced decreases in prostaglandin production and prostaglandin synthase activity

The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content in tissue cultured, avlan skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the effects of Dex and mechanical stretch on prostaglandin production and prostaglandin H synthase (PGHS) activity were examined. In static cultures, 10^?8 M Dex reduced PGF_2α production 55–65% and PGE₂ production 84–90% after 24–72 h of incubation. Repetitive 10% stretch-relaxations of non-Dex-treated cultures increased PGF_2α efflux 41% at 24 h and 276% at 72 h, and increased PGE₂ production 51% at 24 h and 236% at 72 h. Mechanical stimulation of Dex-treated cultures increased PGF_2α production 162% after 24 h, returning PGF_2α efflux to the level of non-Dex-treated cultures. At 72 h, stretch increased PGF_2α efflux 65% in Dex-treated cultures. Mechanical stimulation of Dex-treated cultures also increased PGE₂ production at 24 h, but not at 72 h. Dex reduced PGHS activity in the muscle cultures by 70% after 8–24 h of incubation, and mechanical stimulation of the Dex-treated cultures increased PGHS activity by 98% after 24 h. Repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by mitigating the Dex-induced declines in PGHS activity and prostaglandin production. © 1994 wiley-Liss, Inc.     

Glutamate formation by a new In vitro enzyme system consisting of purified glutamine synthetase and glutamate synthase

After the addition of ammonia to the culture medium, the concentration of glutamine in B. flavum cells increased in 20 s with a decrease in glutamate. In the subsequent 30 s, the glutamine concentration deceased again with an increase in glutamate. An enzyme system, which consisted of purified glutamine synthetase (GS) and glutamate synthase (GOGAT) with ATP- and NADPH-regenerating systems, was made up to study the functions of the GS/GOGAT pathway: concentrations of the substrates and of the enzymes were decided on according to the intracellular conditions. Changes in the concentrations of amino acids caused by the addition of ammonia to the system were very similar to those of intracellular glutamate and glutamine when ammonia was added to the bacterial culture. The time required for the complete formation of glutamate from 0.5 mM ammonia was about 4-times shorter in the GS/GOGAT system than in the system using purified glutamate dehydrogenase (GDH) and the NADPH-regenerating system. The glutamate synthase reaction in the GS/GOGAT system was inhibited by some amino acids much more markedly than in the standard assay mixture consisting of glutamine, α-ketoglutarate and NADPH. These results gave further evidence elucidating the operation of the GS/GOGAT pathway in ammonia assimilation, and suggested that a reconstructed enzyme system is useful for studying physiological mechanisms.     

Evidence for a free radical mechanism of styrene-glutathione conjugate formation catalyzed by prostaglandin H synthase and horseradish peroxidase

We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H-labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes.     

Cooxidation of benzidine by horseradish peroxidase and subsequent formation of possible thioether conjugates of benzidine

The human bladder carcinogen BENZIDINE was found to be converted by horseradish peroxidase and H₂O₂ to an intermediate which reacts with thiol-containing nucleophiles to give rise to a pattern of products which are identical to those produced by reaction of the two electron oxidation product of benzidine, 4,4′-biphenoquinonediimine, under identical conditions at physiological pH. The properties of the major reaction product are dependent on the identity of the thiol employed and are consistent with those expected for a ring-(S)-thioether conjugate of benzidine. These results may be indicative of a previously unrecognized minor pathway of benzidine metabolism.     

Different metabolism by mammalian and horseradish peroxidases in vitro of steroidal estrogens and their catechol metabolites

Radioactively labeled estradiol-17 beta and 17 alpha-ethynylestradiol and their 2-hydroxy derivatives were incubated with either horseradish peroxidase or mouse uterus peroxidase, and the formation of polar products, which could not be extracted with diethyl ether, and of ether-extractable metabolites was studied. Moreover, the extent of DNA binding was determined. The different peroxidases gave rise to different products, indicating that different pathways in the metabolism of these steroidal estrogens are catalysed by the two peroxidases.     

Separation of steroidal estrogens and their major unconjugated metabolites by high performance liquid chromatography

A high performance liquid chromatographic method is described for the rapid, non-destructive separation of a number of physiologically important steroidal estrogens, including the labile catechol estrogens. This procedures uses a "Diol" column and gradient elution to separate in a single run, estrogens ranging from 2-methoxy estrone, one of the least polar C18 steroids, to estriol, one of the most polar. Simpler, isocratic conditions, are provided for the separation of estrogens of similar polarity. A semi-preparative column of similar composition was used for the purification of samples containing 25 to 50 mg of individual steroids.     

Bioactivation of xenobiotics by prostaglandin H synthase

Prostaglandin H synthase (PHS) catalyzes the oxidation of arachidonic acid to prostaglandin H2 in reactions which utilize two activities, a cyclooxygenase and a peroxidase. These enzymatic activities generate enzyme- and substrate-derived free radical intermediates which can oxidize xenobiotics to biologically reactive intermediates. As a consequence, in the presence of arachidonic acid or a peroxide source, PHS can bioactivate many chemical carcinogens to their ultimate mutagenic and carcinogenic forms. In general, PHS-dependent bioactivation is most important in extrahepatic tissues with low monooxygenase activity such as the urinary bladder, renal medulla, skin and lung. Mutagenicity assays are useful in the detection of compounds which are converted to genotoxic metabolites during PHS oxidation. In addition, the oxidation of xenobiotics by PHS often form metabolites or adducts to cellular macromolecules which are specific for peroxidase- or peroxyl radical-dependent reactions. These specific metabolites and/or adducts have served as biological markers of xenobiotic bioactivation by PHS in certain tissues. Evidence is presented which supports a role for PHS in the bioactivation of several polycyclic aromatic hydrocarbons and aromatic amines, two classes of carcinogens which induce extrahepatic neoplasia. It should be emphasized that the toxicities induced by PHS-dependent bioactivation of xenobiotics are not limited to carcinogenicity. Examples are given which demonstrate a role for PHS in pulmonary toxicity, teratogenicity, nephrotoxicity and myelotoxicity.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Specific mixed disulfide formation with purified bovine cardiac glycogen synthase I and glutathione

Bovine cardiac glycogen-free glycogen synthase I reacts with oxidized glutathione at low temperature to partially inactivate the enzyme. Evidence is presented that a mixed disulfide between glutathione and the enzyme is formed in this reaction. A short incubation of the GSSG-treated enzyme with dithiothreitol restores full enzyme activity. The reaction with GSSG is pH dependent and the product is quite stable at neutral pH. Oxidation of one sulfhydryl group in glycogen synthase is associated with a loss of 60-70% of the enzyme activity. Further modification of protein sulfhydryls has less effect on the enzyme activity. Other low molecular weight disulfides also inactivate glycogen synthase and treatment with [35S]cystine to produce a 40% loss of enzyme activity gave rise to a single major radioactive peptide after cyanogen bromide digestion. Thus the GSSG-mediated inactivation of glycogen synthase apparently occurs through a single reactive sulfhydryl group that forms a mixed disulfide with low molecular weight disulfide molecules. Uridine 5'-diphosphate glucose and glycogen prevent the inactivation of glycogen-free glycogen synthase with GSSG, and glucose 6-phosphate retards the rate of inactivation. Reduction and reactivation of the GSSG-oxidized glycogen synthase is not affected by glycogen and it occurs readily at neutral pH with dithiothreitol, mercaptoethanol, or cysteamine. Oxidation of the reactive sulfhydryl group with GSSG has no effect on the rate of glycogen synthase phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.     

The oxidation of arachidonic acid by the cyclooxygenase activity of purified prostaglandin H synthase: spin trapping of a carbon-centered free radical intermediate

The ESR spin trapping technique was used to study the first detectable radical intermediate in the oxidation of arachidonic acid by purified prostaglandin H synthase. The holoenzyme and the apoenzyme, reconstituted with either hematin or Mn2+ protoporphyrin IX, were investigated. Depending on the different types of enzyme activity present, arachidonic acid was oxidized to at least two free radicals. One of these radicals is thought to be the first ESR detectable radical intermediate in the conversion of arachidonic acid to prostaglandin G2 and was detected previously in incubations of ram seminal vesicle microsomes, which are rich in prostaglandin H synthase. The ESR findings correlated with oxygen incorporation into arachidonic acid and prostaglandin formation, where the spin trap inhibits oxygen incorporation and prostaglandin formation by apparently competing with oxygen for the carbon-centered radical. Substitution of arachidonic acid by octadeuterated (5, 6, 8, 9, 11, 12, 14, 15)-arachidonic acid confirmed that the radical adduct contained arachidonic acid that is bound to the spin trap at one of these eight positions. An attempt was made to explain the apparent time lag between the metabolic activity observed in the oxygraph measurements and the appearance of the trapped radical signals.     

Activation of aromatic amines by prostaglandin H synthase

Prostaglandin H synthase catalyzes the first step in the synthesis of prostaglandins from arachidonic acid. The peroxidase activity of this enzyme can support the oxidation of xenobiotics, particularly aromatic amines. This pathway of metabolism may contribute to the activation of carcinogenic aromatic amines in target tissues such as the skin, lung, and bladder. In this review, recent work on this subject is summarized. I emphasize the elucidation of the structures of aromatic amine oxidation products, and their interactions with biological macromolecules. Prostaglandin H synthase supports the activation of benzidine to a mutagenic species in the Ames (Salmonella typhimurium) test, and our studies of the mechanism of this activation are described.