Estrogenic and neuroprotective activity of the neurosteroid dehydroepiandrosterone

The aim of the study was triple: (1) To determine whether dehydroepiandrosterone (DHEA) exerts hormonal activity in breast cancer cells (MCF-7), in endometrial adenocarcinoma cells (Ishikawa) as well as in prostate adenocarcinoma cells (LNCaP). We were also interested in the type of the receptor which is being involved in mediating DHEA’s activity. The results indicate that DHEA exhibits estrogenic activity in Ishikawa adenocarcinoma cells and in MCF-7 breast cancer cells while it is unable to induce proliferation in LNCaP prostate cells. DHEA activation of MCF-7 cell proliferation was mediated through ERα and required mainly conversion of DHEA to 5-androstenediol (5-diol). Given that 17β hydroxy-steroid-dehydrogenase type 1 (17β HSD-1), the enzyme that converts DHEA to 5-diol, is overexpressed in breast cancer cells, the use of DHEA as a dietary supplement is contraindicated in high risk subjects for breast cancer. (2) To study whether DHEA can modulate the anticancer activity of a chemopreventive factor (ebenfuran III), which is being uptaken by the diet. The results showed that estradiol, at physiological concentrations, was able to improve the anticancer activity of ebenfuran III in breast cancer cells, while DHEA, even at supraphysiological concentrations, was ineffective in this respect. (3) To study whether DHEA and its metabolites can protect the immortalized hippocampal HT22 cells from oxidative stress-induced cell death. The results showed that only E2 is able to protect the HT22 cells, in pharmacological concentrations, while DHEA is ineffective in this respect. We also showed that estradiol, at physiological concentrations, was able through ERα to improve the neuroprotective potency of a synthetic antioxidant (1,4 benzoxazine).O σκοπός της διατριβής ήταν τριπλός: (1) Να διευκρινιστεί εάν η δεϋδροεπιανδροστερόνη (DHEA) έχει ορμονική δράση σε καρκινικά κύτταρα μαστού (MCF-7), μήτρας (Ishikawa) και προστάτη (LNCaP) και μέσω ποιου υποδοχέα εξασκεί τη δράση της. Τα αποτελέσματα έδειξαν ότι η DHEA παρουσιάζει οιστρογονική δράση σε καρκινικά κύτταρα μαστού και μήτρας ενώ δεν επάγει τον πολλαπλασιασμό στα κύτταρα αδενοκαρκινώματος του προστάτη. Ειδικά για την επαγωγή του πολλαπλασιασμού στα κύτταρα MCF-7, η δράση αυτή μεσολαβείται από τον υποδοχέα οιστρογόνων τύπου α (ERα) και οφείλεται κυρίως στο μεταβολισμό της DHEA σε 5-ανδροστενοδιόλη (5-diol). Δεδομένου ότι η 17β υδροξυ-στεροειδο-δεϋδρογενάση τύπου 1 (17β HSD-1), που είναι το ένζυμο που μετατρέπει την DHEA σε 5-diol, υπερεκφράζεται στον καρκινικό ιστό, η χρήση της DHEA αντενδείκνυται σε άτομα υψηλού κινδύνου για καρκίνο του μαστού. (2) Να μελετηθεί η δράση της DHEA σε συνδυασμό με ένα χημειοπροστατευτικό παράγοντα (την εμπενφουράνη ΙΙΙ) που προσλαμβάνεται με συγκεκριμένη διατροφή. Τα αποτελέσματα έδειξαν ότι η οιστραδιόλη σε φυσιολογικές συγκεντρώσεις ενισχύει την αντικαρκινική δράση της εμπενφουράνης ΙΙΙ σε καρκινικά κύτταρα του μαστού. Αντίθετα η DHEA, ακόμη και σε πολύ υψηλές συγκεντρώσεις, δεν βελτιώνει την αντικαρκινική δράση της εμπενφουράνης ΙΙΙ. (3) Να μελετηθεί η νευροπροστατευτική δράση της DHEA και των μεταβολιτών της στα νευρικά κύτταρα ΗΤ22 όταν αυτά υπόκεινται σε οξειδωτικό στρες. Το αποτέλεσμα της μελέτης αυτής έδειξε ότι μόνο η οιστραδιόλη προστατεύει τα κύτταρα ΗΤ22 από το θάνατο λόγω οξειδωτικού στρες, σε φαρμακολογικές συγκεντρώσεις, ενώ η DHEA όχι. Επίσης η οιστραδιόλη, δρώντας μέσω του ERα σε φυσιολογικές συγκεντρώσεις, βελτιώνει τη νευροπροστατευτική ικανότητα ενός συνθετικού αντιοξειδωτικού (1,4 βενζοξαζίνη)

The effect of CMGE and caffeine on the expression levels of <i>Cyp1a1</i> and <i>Cyp1a2</i> mRNAs in rat liver.

<p>The level of mRNA expression (normalized to β-actin) is presented relative to that in animals treated with the vehicle only. Each value represents the mean ± SEM of at least three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Games-Howell post test, **, p<0.01.</p

Modulation of CYP1A1 and CYP1A2 Hepatic Enzymes after Oral Administration of <i>Chios Mastic Gum</i> to Male Wistar Rats

<div><p><i>Chios mastic gum</i> (CMG), a resin derived from <i>Pistacia lentiscus</i> var. <i>chia</i>, is known since ancient times for its pharmacological activities. CYP1A1 and CYP1A2 enzymes are among the most involved in the biotransformation of chemicals and the metabolic activation of pro-carcinogens. Previous studies referring to the modulation of these enzymes by CMG have revealed findings of unclear biological and toxicological significance. For this purpose, the modulation of CYP1A1 and CYP1A2 enzymes in the liver of male Wistar rats following oral administration of CMG extract (CMGE), at the levels of mRNA and CYP1A1 enzyme activity, was compared to respective enzyme modulation following oral administration of a well-known bioactive natural product, caffeine, as control compound known to involve hepatic enzymes in its metabolism. mRNA levels of <i>Cyp1a1</i> and <i>Cyp1a2</i> were measured by reverse transcription real-time polymerase chain reaction and their relative quantification was calculated. CYP1A1 enzyme induction was measured through the activity of ethoxyresorufin-<i>O-</i>deethylase (EROD). The results indicated that administration of CMGE at the recommended pharmaceutical dose does not induce significant transcriptional modulation of <i>Cyp1a1/2</i> and subsequent enzyme activity induction of CYP1A1 while effects of the same order of magnitude were observed in the same test system following the administration of caffeine at the mean daily consumed levels. The outcome of this study further confirms the lack of any toxicological or biological significance of the specific findings on liver following the administration of CMGE.</p></div

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part II: Endocrine-related gene expression assessment on male offspring rat testes

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy and early development can lead to adverse developmental outcomes in offspring. One of the endpoints of concern is feminization. The present study aimed to investigate for any possible correlations with endocrine sensitive parameters in the testes of male rat offspring following dam exposure to three EDCs by assessing the expression of endocrine-related genes. Dienestrol (DIES) [0.37–6.25 μg/kg bw/day], linuron (LIN) [1.5–50 mg/kg bw/day], flutamide (FLU) [3.5–50 mg/kg bw/day] as well as their binary mixtures were administered to sexually mature female rats from gestation day (GD) 6 until postnatal day (PND) 21. Gene expression analysis of Star, Cyp11a1, Cyp17a1, Hsd3b2, Pgr and Insl3 was performed by RT-qPCR. Administration of the anti-androgen FLU alone significantly upregulated Cyp11a1 and Cyp17a1 gene expression while administration of LIN and DIES alone did not alter significantly gene expression. The effects of the binary mixtures on gene expression were not as marked as those seen after single compound administrations. Deregulation of Cyp17a1 in rat pup testis, following administration of FLU alone or in mixtures to dams, was significantly correlated with the observed feminization endpoints in male pups.</p

The effect of CMGE and caffeine on the expression levels of <i>Cyp1a1</i> and <i>Cyp1a2</i> mRNAs in rat liver.

<p>The level of mRNA expression (normalized to β-actin) is presented relative to that in animals treated with the vehicle only. Each value represents the mean ± SEM of at least three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Games-Howell post test, **, p<0.01.</p

The effect of CMGE and caffeine on the activity levels of CYP1A1 in rat liver.

<p>Each value represents the mean ± SEM of at least four samples. Statistical significance was assessed by Kruskal-Wallis H Test followed by Mann-Whitney U test. **:p<0.01</p

Investigating the in vitro steatotic mixture effects of similarly and dissimilarly acting test compounds using an adverse outcome pathway-based approach

Within the EuroMix project, we have previously developed an adverse outcome pathway (AOP)-based in vitro assay toolbox to investigate the combined effects of liver steatosis-inducing compounds in human HepaRG hepatocarcinoma cells. In this study, we applied the toolbox to further investigate mixture effects of combinations, featuring either similarly acting or dissimilarly acting substances. The valproic acid structural analogs 2-propylheptanoic acid (PHP) and 2-propylhexanoic acid (PHX) were chosen for establishing mixtures of similarly acting substances, while a combination with the pesticidal active substance clothianidin (CTD) was chosen for establishing mixtures of dissimilarly acting compounds. We first determined relative potency factors (RPFs) for each compound based on triglyceride accumulation results. Thereafter, equipotent mixtures were tested for nuclear receptor activation in transfected HepG2 cells, while gene expression and triglyceride accumulation were investigated in HepaRG cells, following the proposed AOP for liver steatosis. Dose addition was observed for all combinations and endpoints tested, indicating the validity of the additivity assumption also in the case of the tested mixtures of dissimilarly acting substances. Gene expression results indicate that the existing steatosis AOP can still be refined with respect to the early key event (KE) of gene expression, in order to reflect the diversity of molecular mechanisms underlying the adverse outcome

Sequence of primers used in real-time PCR, amplicon sizes and annealing temperatures.

<p>Sequence of primers used in real-time PCR, amplicon sizes and annealing temperatures.</p

New hydroxystilbenoid derivatives endowed with neuroprotective activity and devoid of interference with estrogen and aryl hydrocarbon receptor-mediated transcription

We have synthesized a series of new (E) stilbenoid derivatives containing hydroxy groups at ring positions identical or similar to those of trans-resveratrol and bearing one or two bulky electron donating groups ortho to 4′-OH and we have evaluated their neuroprotective activity using glutamate-challenged HT22 hippocampal neurons to model oxidative stress-induced neuronal cell death. The most active derivatives, 5-{(E)-2-[3,5-bis(1-ethylpropyl)-4-hydroxyphenyl]ethenyl}-1,3-benzenediol (2), 5-[(E)-2-(3,5-di-tert-butyl-4-hydroxyphenylethenyl)]-1,3-benzenediol (4) and 5-{(1E,3E)-4-[3,5-bis(1-ethylpropyl)-4-hydroxyphenyl]-1,3-butadienyl}-1,3-benzenediol (6), had EC50 values of 30, 45 and 12 nM, respectively, and were ca. 100 to 400-fold more potent than resveratrol. Derivatives 2, 4 and 6 lacked cytotoxic activity against HT22 cells and estrogen receptor agonist or antagonist activity in estrogen response element-dependent gene expression and in estrogen-dependent proliferation of MCF-7 human breast cancer cells. In addition, they were incapable of interfering with aryl hydrocarbon receptor-mediated xenobiotic response element-dependent gene expression. Derivatives 2, 4 and 6 might assist in the development of lead candidates against oxidative stress-driven neurodegenerative diseases that will not increase endocrine cancer risk nor affect drug activation and detoxification mechanisms

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part I: Feminization effects on male rat offspring

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy can result in negative health effects in later generations, including sex changes and feminization. The present study assessed the feminization effects on male offspring rats of three EDCs: Dienestrol (DIES), Linuron (LIN), and Flutamide (FLU). Sexually mature female rats were exposed from gestation day (GD) 6 until postnatal day (PND) 21 to: 0.37, 0.75, 1.5, 3.12 or 6.25 μg/kg/day of DIES, 1.5, 3, 6, 12.5, 25 or 50 mg/kg/day of LIN, 3.5, 6.7, 12.5, 25 or 50 mg/kg/day of FLU, and the following mixtures: FLU + DIES (mg/kg/day+μg/kg/day), 3.5 + 0.37, or 3.5 + 3, 25 + 0.37, or 25 + 3; FLU + LIN (mg/kg/day + mg/kg/day), 3.5 + 12.5, or 25 + 12.5; and DIES + LIN (μg/kg/day + mg/kg/day), 0.37 + 12.5, or 3 + 12.5. Anogenital distance (AGD), nipple retention (NR) and cryptorchidism were evaluated. FLU produced a decrease of AGD, an increase of NR, and an increase of cryptorchidism at the highest dose. None of these three endpoints were significantly affected by LIN or DIES treatments alone. Combinations of FLU + LIN and FLU + DIES increased NR, and decreased AGD, while DIES + LIN did not produce any effects in male pups. Results show that FLU is able to induce feminization in male pups, while binary combinations of LIN and DIES did not modify the effects produced by FLU.</p