Assessing placental corticotrophin-releasing hormone disruption by hexestrol in a cell model.

Studies have shown that corticotrophin-releasing hormone (CRH) and relaxin are associated with early delivery. Our lab previously has shown the mycotoxin zeranol increases placental CRH expression. The mycotoxin is used in the farming industry to promote cattle growth, and some synthetic hormones are also used for the same purposes. In order to complete the picture of these growth promoting agents, we attempted to examine the synthetic hormones on the placental gene expression in the current study. Among the tested compounds, hexestrol induced the CRH mRNA and protein expression at 100nM in JEG-3 cells. As signal transduction pathways have been described in the transcriptional control previously, the activations of several protein kinases were determined. P38, PKCß and JNK were activated upon hexestrol treatment. Since the P38-inhibitor SB203580 prevented hexestrol from inducing CRH in a subsequent experiment, P38 was likely involved in the transcriptional regulation. Electrophoretic mobility shift assay revealed an increase in the CRE binding activity in CRH promoter. This study showed that hexestrol exposure might be a concern for pregnant women.

Hexestrol- and nonylphenol-induced differential vitellogenin synthesis in female and male barfin plaice Liopsetta pinnifasciata.

The effects of hexestrol (HXS) and nonylphenol (NP) on plasma vitellogenin (Vtg) concentration in barfin plaice Liopsetta pinnifasciata was studied during spring and autumn experiment. In L. pinnifasciata two "complete" forms of Vtgs, namely VtgAa and VtgAb, were previously described which may be separated due to molecular mass of their largest polypeptide in SDS-PAGE. In spring, the injection of HXS led to an increase in Vtg concentrations in both females and males. SDS-PAGE analysis of plasma from HXS-exposed fish produced only one prominent band at a molecular mass of 180 kDa that corresponds to an increase in VtgAb levels. NP injected in fish in spring induced statistically significant increasing of Vtg concentration in males, and only one type of Vtg, as in case of HXS, accumulated in plasma. In autumn, the injection of HXS results to the increase of Vtg concentration in the plasma of females and males, electrophoretic analysis of plasma proteins showed that only a 98 kDa polypeptide, corresponding to the VtgAa-type showed a significant increase. The blood plasma ratios of VtgAa and VtgAb in experimental fish are discussed in relation to the season and stage of reproductive cycle.

Steatohepatitis-inducing drugs trigger cytokeratin cross-links in hepatocytes. Possible contribution to Mallory-Denk body formation.

Mallory-Denk bodies (MDB) are hepatocyte inclusions containing cytokeratin 8 (CK8) which can develop, along with other steatohepatitis lesions, in patients treated with amiodarone, perhexiline maleate or 4,4'-diethylaminoethoxyhexestrol. These drugs accumulate lipids, whose subsequent peroxidation liberates reactive by-products, like malondialdehyde (MDA). The formation of MDB has been previously reproduced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine or griseofulvin administration which cross-link CK8 by tissue transglutaminase, thus forming an entangled network, from which MDB progressively arise. The present study depicts the mechanisms initiating MDB formation by steatohepatitis-inducing drugs. Short incubation of hepatocytes with amiodarone (50 microM), 4,4'-diethylaminoethoxyhexestrol (50 microM) or perhexiline maleate (25 microM) increased the pool of CK8 monomers and increased cell calcium to activate Ca(++)-dependent transglutaminases which cross-linked the CK8 monomers into CK8-containing oligomers. The present study also provides the first evidence that MDA might directly participate in MDB formation, as this reactive agent cross-linked purified CK8 or albumin in vitro, disrupted the cytokeratin network of isolated hepatocytes, and bridged CK8 molecules. In conclusion, steatohepatitis-inducing drugs increase cell calcium and activate tissue transglutaminase, which cross-links CK8 to form a molecular scaffold, from which MDB might secondarily arise. Malondialdehyde also cross-links CK8, albeit through a different mechanism, and might also contribute to MDB formation.

Formation of the depurinating N3adenine and N7guanine adducts by reaction of DNA with hexestrol-3',4'-quinone or enzyme-activated 3'-hydroxyhexestrol. Implications for a unifying mechanism of tumor initiation by natural and synthetic estrogens.

The nonsteroidal synthetic estrogen hexestrol (HES), which is diethylstilbestrol hydrogenated at the C-3-C-4 double bond, is carcinogenic. Its major metabolite is the catechol, 3'-OH-HES, which can be metabolically converted to the catechol quinone, HES-3',4'-Q. Study of HES was undertaken with the scope to substantiate evidence that natural catechol estrogen-3,4-quinones are endogenous carcinogenic metabolites. HES-3',4'-Q was previously shown to react with deoxyguanosine to form the depurinating adduct 3'-OH-HES-6'-N7Gua by 1,4-Michael addition [Jan S-T, Devanesan PD, Stack DE, Ramanathan R, Byun J, Gross ML, et al. Metabolic activation and formation of DNAadducts of hexestrol,a synthetic nonsteroidal carcinogenic estrogen. Chem Res Toxicol 1998;11:412-9.]. We report here formation of the depurinating adduct 3'-OH-HES-6'-N3Ade by reaction of HES-3',4'-Q with Ade by 1,4-Michael addition. The structure of the N3Ade adduct was established by NMR and MS. We also report here formation of the depurinating 3'-OH-HES-6'-N7Gua and 3'-OH-HES-6'-N3Ade adducts by reaction of HES-3',4'-Q with DNA or by activation of 3'-OH-HES by tyrosinase, lactoperoxidase, prostaglandin H synthase or 3-methylcholanthrene-induced rat liver microsomes in the presence of DNA. The N3Ade adduct was released instantaneously from DNA, whereas the N7Gua adduct was released with a half-life of approximately 3 h. Much lower (<1%) levels of unidentified stable adducts were detected in the DNA from these reactions. These results are similar to those obtained by reaction of endogenous catechol estrogen-3,4-quinones with DNA. The similarities extend to the instantaneously-depurinating N3Ade adducts and relatively slowly-depurinating N7Gua adducts. The endogenous estrogens, estrone and estradiol, their 4-catechol estrogens and HES are carcinogenic in the kidney of Syrian golden hamsters. These results suggest that estrone (estradiol)-3,4-quinones and HES-3',4'-Q are the ultimate carcinogenic metabolites of the natural and synthetic estrogens, respectively. Reaction of the electrophilic quinones by 1,4-Michael addition with DNA at the nucleophilic N-3 of Ade and N-7 of Gua is suggested to be the major critical step in tumor initiation by these compounds.

Metabolic activation and formation of DNA adducts of hexestrol, a synthetic nonsteroidal carcinogenic estrogen.

Hexestrol (HES), a synthetic nonsteroidal estrogen, is carcinogenic in Syrian golden hamsters. The major metabolite of HES is its catechol, 3'-OH-HES, which can be metabolically converted to the electrophilic catechol quinone, HES-3',4'-Q, by peroxidases and cytochrome P450. Standard adducts were synthesized by reacting HES-3',4'-Q with dG and dA to produce the adducts 3'-OH-HES-6'(alpha, beta)-N7Gua and HES-3',4'-Q-6'-N6dA, respectively. When HES-3',4'-Q was reacted with calf thymus DNA, 3'-OH-HES-6'(alpha,beta)-N7Gua was identified by HPLC and tandem mass spectrometry as the depurinating adduct, with minor amounts of stable adducts. 3'-OH-HES was bound to DNA after activation by horseradish peroxidase, lactoperoxidase, or rat liver microsomes. The depurinating adduct 3'-OH-HES-6'(alpha, beta)-N7Gua was identified in these systems at levels of 65, 41, and 11 micromol/mol of DNA-P, respectively. Unidentified stable adducts were observed in much lower amounts and were quantified by the 32P-postlabeling method. Similarly to 3'-OH-HES, the catechol metabolites of the natural steroidal estrogens estrone (E1) and estradiol (E2), namely, 2-OHE1, 4-OHE1, 2-OHE2, and 4-OHE2, can be oxidized to their corresponding quinones by peroxidases and cytochrome P450. The quinones of the carcinogenic 4-OHE1 and 4-OHE2 have chemical and biochemical properties similar to those of HES-3',4'-Q. The results suggest that formation of HES-3',4'-Q may be a critical event in tumor initiation by HES and that HES is an excellent model compound to corroborate the hypothesis that estrogen-3,4-quinones are ultimate carcinogenic metabolites of the natural steroidal estrogens E1 and E2.

Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes.

BACKGROUND & AIMS: 4,4'-Diethylaminoethoxyhexestrol (DEAEH), amiodarone, and perhexiline cause steatohepatitis in humans. The mechanisms of these effects are unknown for DEAEH and have not been completely elucidated for amiodarone and perhexiline. The aim of this study was to determine these mechanisms. METHODS: Rat liver mitochondria, cultured rat hepatocytes, or rats were treated with these drugs, and the effects on mitochondrial respiration, beta-oxidation, reactive oxygen species formation, and lipid peroxidation were determined. RESULTS: DEAEH accumulated in mitochondria and inhibited carnitine palmitoyl transferase I and acyl-coenzyme A dehydrogenases; it decreased beta-oxidation and caused lipid deposits in hepatocytes. DEAEH also inhibited mitochondrial respiration and decreased adenosine triphosphate (ATP) levels in hepatocytes. DEAEH, amiodarone, and perhexiline augmented the mitochondrial formation of reactive oxygen species and caused lipid peroxidation in rats. CONCLUSIONS: Like amiodarone and perhexiline, DEAEH accumulates in mitochondria, where it inhibits both beta-oxidation (causing steatosis) and respiration. Inhibition of respiration decreases ATP and also increases the mitochondrial formation of reactive oxygen species. The latter oxidize fat deposits, causing lipid peroxidation. We suggest that ATP depletion and lipid peroxidation may cause cell death and that lipid peroxidation products may account, in part, for other steatohepatitis lesions.

Lysosomal storage of sulphated glycosaminoglycans induced by dicationic amphiphilic drug molecules: significance of the central planar ring system.

The immunomodulatory drug tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) and several congeners are known to disturb the lysosomal degradation of sulphated glycosaminoglycans and thereby induce lysosomal storage of glycosaminoglycans in cultured cells and intact organisms. The molecules of tilorone and congeners consist of a planar aromatic ring system symmetrically substituted with two aliphatic side chains each carrying a protonizable nitrogen. In a previous study it was proposed that non-degradable glycosaminoglycan-drug complexes are formed by electrostatic interactions and that additionally intermolecular interactions between the drug molecules due to electronic coupling of their central planar ring system are important for formation and stabilization of the glycosaminoglycan-drug complexes and thus for the drug side effect in question. The significance of the central planar ring system was tested in the present study by comparing tilorone and the compound bis(beta-diethylamino-ethylether)hexestrol (DH) with respect to their potencies to cause lysosomal glycosaminoglycan storage in cultured bovine corneal fibroblasts. DH has the same side chains as tilorone, but its central apolar moiety lacks planarity. At a concentration (1.75 muM) which did not cause enhanced secretion of the lysosomal enzyme beta-hexosaminidase (E.C. 3.2.1.52), DH was significantly less potent than tilorone in causing storage of [35S]glycosaminoglycans. This is taken as support of the hypothesis that the planar tricyclic ring system is essential for the high potency of tilorone and its congeners to exert this adverse action.

Pituitary-related weight changes affecting the liver, uterus and adrenal glands of rats treated with hexoestrol and clomiphene in high doses.

The synthetic oestrogen hexoestrol, administered at 60 mg/kg/day for 4 days to female rats in 3 studies, caused the following mean changes in the relative weights of some of the principal organs: liver (+37%), spleen (-11%), adrenals (+43%), kidneys (+3%), pituitary (+23%), uterus (+49%), and ovaries (+13%). The heart weights showed no consistent changes. The mean relative organ weights of hypophysectomized, hexoestrol-treated rats did not differ significantly from those of untreated hypophysectomized controls. The latter animals had lower organ weights than sham-operated controls. Pretreatment with clomiphene citrate at dose levels of 20-60 mg/kg/day prevented in a dose-dependent manner most of the organ weight changes induced by hexoestrol. The exception was the adrenal weight, which was increased. Compared with controls, the mean relative organ weights of rats receiving clomiphene alone at 60 mg/kg/day differed as follows: liver (-18%), spleen (-17%), heart (-16%), adrenals (+7%), kidneys (-11%), pituitary (-13%), uterus (-25%), and ovaries (-4%). The changes affecting the liver, spleen, heart, kidneys and uterus were significant. Rats immunised with a monkey antiserum to rat growth hormone and treated with hexoestrol had significantly lower relative liver weights than did animals treated with hexoestrol alone. No other significant differences were observed. It is tentatively concluded that there is a mediating or co-operative pituitary influence involved in the significant hepatic, uterine and adrenal weight gains caused by hexoestrol. Clomiphene may act centrally to affect these organ weight changes and, indeed, may act at this level to (mainly) anti-organotrophic effect even in the absence of exogenous oestrogen. In the case of the hexoestrol-induced liver enlargement, the role of pituitary growth hormone is worth further investigation.

Morphology and anatomic localization of renal microneoplasms and proximal tubule dysplasias induced by four different estrogens in the hamster.

Estrogens are known to induce tumors in high frequency in orchiectomized Syrian golden hamsters, but the histogenesis of the tumors is controversial. In order to identify the earliest precursors of the tumors, animals were implanted with pellets of four different estrogens, sacrificed at times ranging up to 6.4 months, and the various neoplastic and nonneoplastic lesions were characterized. Infiltrating cancers were identified in 80% of animals exposed to diethylstilbestrol, 17 beta-estradiol, and hexestrol for periods of 5.3-6.4 months; however hamsters exposed to ethinyl estradiol for comparable times did not develop any tumors. Proximal tubule dysplasia, identified as focal collections of abnormal-appearing cells with increased [3H]thymidine-labeling indices (eight times higher than nondysplastic cells), was the only nonmalignant change that, for every agent, either preceded or accompanied the development of cancer. The dysplastic lesions were further subdivided into two types when it became apparent that they and carcinoma in situ, another lesion in the proximal tubules, might be part of a continuum of tumor progression that results in infiltrating cancer. Another dysplastic variant, classified as florid dysplasia because of its extensive involvement of tubules, showed well-differentiated features; it was seen only in the ethinyl estradiol-treated hamsters. In a quantitative study of the anatomic localization of dysplasias and microcancers (less than 0.5 mm in diameter) induced by diethylstilbestrol, both lesions showed highest incidence in the deep renal parenchyma. The dysplasias were at least eight times more prevalent in the proximal tubules of the innermost 10% of the cortex and subjacent medulla than in the rest of the cortex. We conclude that proximal tubule dysplasias developing in the deep renal parenchyma are the most likely precursors of the estrogen-induced cancers.

Antiestrogen and antiandrogen administration reduce bone mass in the rat.

In mature female and male rats sex hormone deficiency was produced by surgical castration and by antiestrogen or antiandrogen administration. For the latter purpose we used the nonsteroidal antiestrogens tamoxifen, keoxifene (LY156758) and tetramethylhexestrol, and the steroidal antiandrogen cyproterone acetate. Dosages of 0.4 mg tamoxifen/rat/day and isomolar dosages of keoxifene and tetramethylhexestrol led to a bone mass reduction which was comparable to ovariectomized rats. Cyproterone acetate showed, at 10 mg/rat/day, a similar decrease in bone mass like orchidectomy. The often discussed intrinsic estrogen activity of the antiestrogens was present only in the highest dosage tested of tamoxifen. Keoxifene and tetramethylhexestrol showed no estrogenic effects, but this may be a dosage problem. Cyproterone acetate revealed no androgenic side-effects. These results indicate that antigonadal hormone drugs reduce bone mass to a varying extent.

Subacute toxicity of diethylstilboestrol and hexoestrol in the female rat, and the effects of clomiphene pretreatment.

Two groups, each of ten female rats, were orally dosed with the synthetic oestrogens diethylstilboestrol (DES; 6 mg/kg day) and hexoestrol (60 mg/kg/day) for 6 wk. A further group of ten animals received clomiphene citrate (2 mg/kg/day) and DES (6 mg/kg/day), while two groups, each of ten animals, acted as controls. One animal treated with DES died within the 42-day study period. Its death was associated with a bleeding disorder related to severe liver damage. The remaining treated animals showed little overt toxicity. The major haematological finding in the treated animals that survived was a moderate, stable, normocytic, normochromic anaemia. The terminal bone marrows of these animals showed a modest degree of erythroid hypoplasia. There were stimulatory and other changes in the reproductive tract and there were gains in the liver, adrenal and pituitary weights. These effects were more marked in the rats treated with hexoestrol, the gains in the relative organ weights being particularly striking: liver, 51%, adrenals, 210%, uterus, 79% and pituitary, 500%. The oestrogens caused reductions in body weight (18% in both cases) and appetite, a modest degree of fatty change in the liver, and alterations in the serum proteins. The Thrombotest clotting times of the hexoestrol-treated rats were prolonged. Clomiphene citrate decreased the adverse effects of DES on appetite and body weight, and sharply decreased the gains in the pituitary and uterine weights from 243 to 80% and 59 to 14%, respectively. The toxic effects of the oestrogens in the rat were more closely related to the dose administered than to their hormonal potency. Oestrogens are less toxic to the rat than they are to the cat, dog and ferret, and the toxic effects are in many respects qualitatively different.

Hepatotoxicity of the synthetic oestrogen hexoestrol in the female rat.

The synthetic oestrogen hexoestol is hepatotoxic to the female rat. Twenty-five animals were treated daily with hexoestrol at a dose of 60 mg/kg. Twenty-five more acted as controls. Five animals from each group were killed after 4, 12, 20, 30 and 40 days of treatment to permit serial evaluations of liver histopathology to complement serum biochemical investigations. There were no mortalities during the study and only modest external signs of toxicity. All the treated rats showed reductions in body weight and appetite and gains in liver weight. This latter effect was due to both cellular hypertrophy and hyperplasia. The principle finding of the liver histopathology was fatty change. This affected scattered individual cells, mainly in the midzonal region. All of the serum parameters of toxicity--which consisted of bilirubin, total protein, albumin, glycoprotein, alpha 2-macrofoetoprotein, and alkaline phosphatase--indicated liver impairment. Most of these also showed time-related changes consistent with an initial phase up to Day 20 of marked liver dysfunction superseded by a more adaptive phase thereafter. The hepatotoxicity described here seems sufficient to explain blood clotting defects observed elsewhere in oestrogen-treated rats.

High incidence of hepatocellular carcinomas after synthetic estrogen administration in Syrian golden hamsters fed alpha-naphthoflavone: a new tumor model.

P8 80-100% incidence of multinodular hepatocellular carcinomas was observed in castrated male hamsters following synthetic estrogen treatment in the presence of 0.2-0.4% alpha-naphthoflavone (ANF) in the diet after 8.5-10 months. Induction of these liver tumors was detected as early as 3.5-4.0 months in low frequency. Of the synthetic estrogens studied, ethynylestradiol (CAS: 57-63-6) was a more potent inducer of these hepatic carcinomas than either diethylstilbestrol (CAS: 56-53-1) or hexestrol (CAS: 84-16-2). ANF, considered an inhibitor of P450-dependent multisubstrate monooxygenases, did not produce any liver tumors when administered alone for up to 12 months. Neither concomitant androgen nor progesterone (CAS: 57-83-0) treatment resulted in any hepatic carcinomas in animals maintained on ANF. Moreover, beta-naphthoflavone (CAS: 6051-87-2) treatment alone or in combination with these synthetic estrogens also resulted in no hepatic tumors. This new estrogen-induced liver tumor model could be useful to elucidate the casual relationship that exists between estrogenic hormones and hepatic tumors in humans.

Ring-substituted 1,2-dialkylated 1,2-bis(hydroxyphenyl)ethanes. 3. Synthesis, estrogen receptor binding affinity, and evaluation of antiestrogenic and mammary tumor inhibiting activity of 2,2'-disubstituted butestrols and 6,6'-disubstituted metabutestrols.

The synthesis of symmetrically 2,2'-disubstituted butestrols [meso-2,3-bis(4-hydroxyphenyl)butanes] and of 6,6'-disubstituted metabutestrols [meso-2,3-bis(3-hydroxyphenyl)butanes] are described [2,2'-substituents: H (1), OH (2), F (3), Cl (4), Br (5), CH3 (6), and C2H5 (7); 6,6'-substituents: H (8), OH (9), Cl (10), and CH3 (11)]. Compounds 1-11 were obtained by reductive coupling of the corresponding 1-phenylethanols with TiCl3/LiAlH4 and separation of the meso diastereomers. The binding affinity of the test compounds to the calf uterine estrogen receptor was measured relative to that of [3H]estradiol by a competitive binding assay. With the exception of 9, all other compounds showed remarkably high relative binding affinity (RBA) values between 1.0 and 29% that of estradiol. Compounds 3 and 6 (RBA values: 15 and 29), as well as 10 and 11 (1.7 and 5.2), exceeded those of the corresponding unsubstituted compounds 1 and 8 (12 and 1.0). The compounds exhibited strong (3, 4, 6, and 7), moderate (1, 2, and 10), weak (11), or no (8) estrogenic activity in the uterine weight test of the immature mouse. Compounds 1, 2, 8, 10, and 11 showed antiestrogenic activity inhibiting the estrone-stimulated uterine growth (25-35% inhibition). Compound 11 led to a significant inhibition of the tumor growth when tested on the 9,10-dimethyl-1,2-benzanthracene induced, hormone-dependent mammary carcinoma of the Sprague-Dawley rat.

[Embryogenesis and early postnatal development of rats following transplacental sex hormone administration]. / Embriogenez i rannee postnatal'noe razvitie krys pri transplatsentarnom vvedenii polovykh gormonov.

A study was made of the effect of different doses of testosterone propionate, sinestrol, diethylstilbestrol and sigetin administered to rats during the last three days of pregnancy on the embryogenesis and postnatal development of the progeny. It was established that sex hormones (except sigetin) often disturb pregnancy, labor and lactation. They also exert a musculinizing and sterilizing effect on the progeny. Sigetin produces no action on embryogenesis or postnatal development of rats.