Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells

Various pesticides known or suspected to interfere with steroid hormone function were screened in H295R cells for effects on catalytic activity and mRNA expression of aromatase. Dibutyl-, tributyl-, and triphenyltin chloride decreased aromatase and ethoxyresorufin O-deethylase activities concentration dependently (1-300 nM; 24-h exposure). However, these decreases occurred only at cytotoxic concentrations, indicated by decreases in mitochondrial MTT reduction and intracellular neutral red uptake. The organotins did not cause direct inhibition during the catalytic assay (1-1000 nM; 1.5-h exposure). The same was true for p,p'-DDT, and o,p-DDT, and o,p-DDE, which decreased aromatase activity only at cytotoxic concentrations (> or =10 microM; 24-h exposure). p,p'-DDE had no effect on aromatase activity or cell viability at 1 and 10 microM. Various imidazole-like fungicides were aromatase inhibitors. Imazalil and prochloraz were potent mixed inhibitors (K(i)/K(i)(') = 0.04/0.3 and 0.02/0.3 microM, respectively), whereas propiconazole, difenoconazole, and penconazole were less potent competitive inhibitors (K(i) = 1.9, 4.5, and 4.7 microM, respectively). Fenarimol, tebuconazole, and hexaconazole decreased aromatase activity close to cytotoxic concentrations. Vinclozolin, as was shown previously for atrazine, induced aromatase activity and CYP19 mRNA levels about 2.5- and 1.5-fold, respectively. To investigate the mechanism of aromatase induction in H295R cells, the ability of the pesticides to increase intracellular cAMP levels was examined. Vinclozolin (100 microM) and atrazine (30 microM) increased cAMP levels about 1.5-fold above control. Forskolin and isobutyl methylxanthine (IBMX) increased cAMP levels 3 and 1.8-fold, respectively. Time-response curves for cAMP induction and concentration-response curves for aromatase induction by vinclozolin, atrazine, and IBMX were similar, suggesting that the mechanism of aromatase induction by these pesticides is mediated through inhibition of phosphodiesterase activity.     

Inhibition and induction of aromatase (CYP19) activity by brominated flame retardants in H295R human adrenocortical carcinoma cells.

Brominated flame retardants (BFRs) are persistent and ubiquitous chemicals in the environment, and they are found at increasing levels in tissues of wildlife and humans. Previous in vitro studies with the BFR class of polybrominated diphenyl ethers (BDEs) have shown endocrine-disrupting properties. Our study assessed the potential effects of nineteen BDEs, five hydroxylated BDEs (OH-BDEs), one methoxylated BDE (CH(3)O-BDE), tetrabromobisphenol-A (TBBPA), its dibromopropane ether derivative (TBBPA-DBPE), and the brominated phenols/anisols 2,4,6-tribromophenol (TBP), 4-bromophenol (4BP) and 2,4,6-tribromoanisole (TBA) on the catalytic activity of the steroidogenic enzyme aromatase (CYP19) in H295R human adrenocortical carcinoma cells. Effects were studied in the concentration range from 0.5 to 7.5 microM; exposures were for 24 h. Both 6-OH-BDE47 and 6-OH-BDE99 showed an inhibitory effect on aromatase activity at concentrations >2.5 microM and >5 microM, respectively. However, 6-OH-BDE47 also caused a statistically significant increase in cytotoxicity (based on mitochondrial MTT reduction and lactate dehydrogenase-leakage [LDH]) at concentrations >2.5 microM that could explain in part the apparent inhibitory effect on aromatase activity. Compared to 6-OH-BDE47, the methoxy analog (6-CH(3)O-BDE47) did not elicit a cytotoxic effect, whereas significant inhibition of aromatase remained. TBP caused a concentration-dependent induction of aromatase activity between 0.5 and 7.5 microM (with a maximum of 3.8-fold induction at 7.5 microM). This induction was not observed when a OH- group replaced the CH(3)O- group or when bromine atoms adjacent to this OH- group were absent. These in vitro results provide a basis for studies of more detailed structure-activity relationships between these brominated compounds and the modulation of aromatase activity.     

Effects of bisphenol A-related diphenylalkanes on vitellogenin production in male carp (Cyprinus carpio) hepatocytes and aromatase (CYP19) activity in human H295R adrenocortical carcinoma cells

The present study investigated the effects of the known xenoestrogen bisphenol A (BPA) relative to eight BPA-related diphenylalkanes on estrogen receptor (ER)-mediated vitellogenin (vtg) production in hepatocytes from male carp (Cyprinus carpio), and on aromatase (CYP19) activity in the human adrenocortical H295R carcinoma cell line. Of the eight diphenylalkanes, only 4,4'-(hexafluoropropylidene)diphenol (BHF) and 2,2'-bis(4-hydroxy-3-methylphenyl)propane (BPRO) induced vtg, i.e., to a maximum of 3% to 4% (at 100 microM) compared with 8% for BPA relative to the maximum induction by 17beta-estradiol (E2, 1 microM). Bisphenol A diglycidyl ether (BADGE) was a potent antagonist of vtg production with an IC50 of 5.5 microM, virtually 100% inhibition of vtg at 20 microM, and an inhibitive (IC50) potency about one-tenth that of the known ER antagonist tamoxifen (IC50, 0.6 microM). 2,2'-Diallyl bisphenol A, 4,4'-(1,4-phenylene-diisopropylidene)bisphenol, BPRO, and BHF were much less inhibitory with IC50 concentrations of 20-70 microM, and relative potencies of 0.03 and 0.009 with tamoxifen. Bisphenol ethoxylate showed no anti-estrogenicity (up to 100 microM), and 4,4'-isopropylidene-diphenol diacetate was only antagonistic at 100 microM. When comparing the (anti)estrogenic potencies of these bisphenol A analogues/diphenylalkanes, anti-estrogenicity occurred at lower concentrations than estrogenicity. 4,4'-Isopropylidenebis(2,6-dimethylphenol) (IC50, 2.0 microM) reduced E2-induced (EC50, 100 nM) vtg production due to concentration-dependent cytotoxicity as indicated by a parallel decrease in MTT activity and vtg, whereas the remaining diphenylalkanes did not cause any cytotoxicity relative to controls. None of the diphenylalkanes (up to 100 microM) induced EROD activity indicating that concentration-dependent, CYP1A enzyme-mediated metabolism of E2, or any Ah-receptor-mediated interaction with the ER, was not a likely explanation for the observed anti-estrogenic effects. At concentrations as great as 100 microM, none of the diphenylalkanes directly inhibited aromatase (CYP19) activity in H295R cells. Environmental exposure of fish to BPA and related diphenylalkanes, depending on the structure, may pose anti-estrogenic, and to a lesser extent estrogenic, risks to development and reproduction.     

Chemopreventive actions by enterolactone and 13 VIOXX-related lactone derivatives in H295R human adrenocortical carcinoma cells

Cytochrome P450c17 (CYP17) has been linked to various hormone-related diseases, including breast cancer, thus being a potential target for cancer chemoprevention. We studied the naturally occurring phytochemical enterolactone (ENL) and 13 VIOXX^®-related lactone derivatives (CRI-1 to CRI-13) for their effects on CYP17 activity and expression and on cell cycle status in the human H295R adrenocorticocarcinoma cell line. Of the tested compounds, only CRI-3, -7, -10 and -12 showed to be inhibitors of CYP17 activity in H295R cells. This inhibition was not due to decreased mRNA expression, but was apparently caused by post-translational modification of the CYP17 enzyme. The MAPK kinase (MEK) inhibitor PD98059 induced CYP17 activity by 24%, while co-incubation of the CRI-s with PD98059, reduced CYP17 activity even further than the reduction caused by the CRI-s alone. In addition, CRI-3, -7, -10 and -12 arrested the cell cycle in the G(2)/M phase. The structure–activity similarities of the CRI-s with known micro-tubule binding agents strongly suggest that cell cycle arrest is a result of interaction with tubulin. We conclude that the proposed cancer chemopreventive actions of ENL are not mediated through interaction with CYP17 or cell cycle status. Of the VIOXX^®-related lactone derivatives, CRI-7 could prove useful in the prevention of hormone-dependent cancers, such as breast cancer, since in vitro it shows low cytotoxicity, it is a potent inhibitor of CYP17 activity and strong inducer of cell cycle arrest.     

Perfluorinated compounds differentially affect steroidogenesis and viability in the human adrenocortical carcinoma (H295R) in vitro cell assay

Perfluorinated compounds (PFCs) comprise a large class of man-made chemicals of which some are persistent and present throughout the ecosystem. This raises concerns about potential harmful effects of such PFCs on humans and the environment. In order to investigate the effects of potentially harmful PFCs on steroid hormone production, human adrenocortical H295R cells were exposed to three persistent PFCs including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) at six different concentrations (6 nM to 600 μM) for 48 h. Exposure to 600 μM PFOS resulted in a dose-responsive increase in oestradiol as well as a smaller dose-responsive increase in progesterone and testosterone secretion measured using radioimmunoassay. The aromatase activity was not significantly altered by PFOS. Only small changes in hormone secretion were detected following exposure to PFOA and PFNA. Gene expression of CYP11A, quantified using qRT-PCR was decreased by all exposure doses of PFOA, whereas HMGR expression was decreased by 60 nM PFNA. The viability markedly decreased by exposure to 600 μM of PFOA or PFNA, but not PFOS. Flow cytometric analysis demonstrated a significant increase in apoptosis following exposure to PFNA at the highest concentration. We conclude that PFOS is capable of altering steroidogenesis in the H295R in vitro model by a mechanism other than changes in gene expression or activity of aromatase. Additionally, PFCs appear to differentially affect cell viability with induction of cell death via apoptosis at high doses of PFNA.     

Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells.

The human placental JEG-3 and JAR choriocarcinoma cell lines have been used as placental models for the study of aromatase (CYP19) activity and endocrine functions. In the present study, 21 organochlorines (OCs) mediated decreases in aromatase activity and protein and DNA content and increases in the percent lactate dehydrogenase (LDH) leakage in JEG-3 cells. These effects were highly variable among the types of OC and their treatment concentrations. Lowest observed effective concentrations reached 0. 001 microM for several OCs. Aromatase activity decreases and OC-mediated cytotoxicity were related. Thus, it was not possible to clearly assess the capacity of the OCs to modulate aromatase activity. Similar to 1,4-naphthoquinone, the most cytotoxic OCs contained a hydroxyl (4'-OH-2,4,6-trichlorobiphenyl and tris(4-chlorophenyl)methanol) or methylsulfonyl- (3- and 4-MeSO(2)-2, 2',5,5'-tetrachlorobiphenyl and -2,3',4',5-tetrachlorobiphenyl, and 3'- and 4'-MeSO(2)-2,2',3,4,5'-pentachlorobiphenyl and -2,2',4,5, 5'-pentachlorobiphenyl) functional group. Modulation of aromatase activity and LDH leakage were less for 3,3',4,4', 5-pentachlorobiphenyl and benzo[a]pyrene and insignificant for five alkyl-substituted trichloro-dibenzofurans and 2,3,7, 8-tetrachloro-dibenzo-p-dioxin (up to 10 microM). Cytotoxicity-related effects were influenced by the cell density and the presence of 10% fetal calf serum in the medium during compound incubation. Similar cytotoxic effects were observed for the JAR cell line. The involvement of an apoptotic mechanism of cytotoxicity in OC-treated JEG-3 cells was suggested by the binding of APO2.7 (an antibody specific to apoptotic cells), DNA fragmentation, and trypan blue staining. JEG-3 and JAR cells appear too sensitive toward OC-mediated cytotoxicity for use as in vitro bioassays to evaluate the potential modulation of aromatase activity. However, these cell lines may prove useful for examining the capacity of xenobiotics to modulate placental toxicity.     

Inhibition of human aromatase complex (CYP19) by antiepileptic drugs.

Antiepileptic drugs and epilepsy are often associated with sexual disorder in women such as hyperandrogenism, menstrual disorders and ovarian cysts. In children, until puberty, a hormone imbalance may influence many aspects of development, e.g. growth and sexual maturation. The aromatase complex is the enzyme system that converts androgens to estrogens and consequently an inhibition may induce a hormone imbalance. Twelve antiepileptic drugs, used in mono or polytherapy for the treatment of children, were tested for their ability to inhibit aromatase (CYP19) with commercially available microsomes from transfected insect cells using dibenzylfluorescein as substrate. The drugs inhibiting CYP19 were: lamotrigine, oxcarbazepine, tiagabine, phenobarbital, phenytoin, ethosuximide, and valproate. The inhibitory effects (50% reduction in activity compared to enzymes without inhibitor present) were in the range of 1.4-49.7 mM. Carbamazepine, gabapentin, primidone, topiramate and vigabatrin showed no inhibition. Additionally, binary drug combinations were tested to investigate if combination therapy could potentiate the aromatase inhibition. Additive inhibition was seen in combination experiments with valproate and phenobarbital. When adding carbamazepine to a range of valproate concentrations no additional inhibition was seen. The data for some of the AEDs show that side effects on steroid synthesis in humans due to inhibition of aromatase should be considered.     

Effects of 3-MeSO2-DDE and some CYP inhibitors on glucocorticoid steroidogenesis in the H295R human adrenocortical carcinoma cell line.

The formation of steroids in the H295R human adrenocortical carcinoma cell line was analysed by HPLC or RIA, and based on these data the apparent catalytic activities of CYP11A, CYP17, CYP21 and CYP11B1 in this cell line were calculated. The environmental pollutant 3-methylsulfonyl-DDE (3-MeSO2-DDE) and the cytochrome P450 (CYP) inhibitors ketoconazole, metyrapone and aminoglutethimide were studied for their effects on the steroid formation. Metyrapone (IC50) of 1 microM) and 3-MeSO2-DDE (10 microM: 66 +/- 10% of control) were found to inhibit the apparent CYP11B1 activity. Ketoconazole inhibited all enzymes examined with the greatest effects on CYP11B1 (IC50) of 2.5 microM). Aminoglutethimide was examined only for effects on CYP11A activity and was shown to inhibit pregnenolone formation (20 microM: 61 +/- 4% of control). The possibility of studying all CYP enzymes in the corticosteroidogenesis makes this cell line a valuable test system to examine effects of chemicals, such as suspected endocrine disruptors, on the human glucocorticoid hormone synthesis. The inhibition of cortisol formation by 3-MeSO2-DDE supports an interaction with the active site of CYP11B1, as previously reported in mouse adrenocortical Y1 cells. In mice, this interaction led to metabolic activation and a high adrenotoxicity of 3-MeSO2-DDE. Therefore studies on the adrenotoxicity of 3-MeSO2-DDE in humans are needed.     

Effect of phthalate esters and alkylphenols on steroidogenesis in human adrenocortical H295R cells

We investigated the inhibitory effects of phthalate esters and alkylphenols on steroidogenesis by human adrenocortical H259R cells, a model of human steroidogenic cells. Dicyclohexyl phthalate (DCHP) at a concentration of 30 μM produced a significant decrease in the dibutyryl cAMP-stimulated cortisol secretion (76% reduction). 4-t-Pentylphenol (4-t-PP), 4-t-octylphenol (4-t-OP) and 4-nonylphenol (4-NP) also produced significant decreases in the dibutyryl cAMP-stimulated cortisol secretion by 58, 34 and 40%, respectively at 50 μM. Reductions in cortisol secretion by these chemicals were dose-dependent. To elucidate the inhibitory effects of DCHP, 4-t-PP, 4-t-OP and 4-NP on cortisol secretion from H295R cells, the effects of these chemicals on various steroidogenic enzymes, such as C(20,22)-lyase (CYP11A), 3β-hydroxysteroid dehydrogenase type II (3β-HSDII), 17α-hyroxylase/C(17,20)-lyase (CYP17), 21-hydroxylase (CYP21B) and 11β-hydroxylase (CYP11B1), were investigated. DCHP significantly inhibited CYP21B activity at 25 μM. 4-t-OP strongly inhibited CYP11A activity at 12.5 and 25 μM, and inhibited CYP17 and CYP21B at 25 μM. Similarly, 4-NP inhibited CYP11A at 25 μM and strongly inhibited CYP17 and CYP21B at 12.5 and 25 μM. Neither DCHP nor any of the alkylphenols tested altered 3β-HSDII activity.     

Serum dioxin-like compounds and aromatase (CYP19) expression in endometriotic tissues

Dioxin-like compounds (DLCs) are suspected etiological factors of endometriosis but their potential mechanisms of action remain elusive. Because endometriosis is an estrogen-dependent disease and since aromatase (CYP19), a key enzyme in estrogen biosynthesis, was recently demonstrated to be expressed in endometriotic lesions, we hypothesized that dioxin-like compounds could modulate local estrogen production through an up-regulation of aromatase. We tested this hypothesis by examining the correlation between serum DLC levels and CYP19 expression in endometriotic tissue obtained from 47 patients with peritoneal, ovarian endometriosis and/or deep endometriotic nodules of the rectovaginal septum. Aromatase expression was assessed by real-time RT-PCR in biopsied endometriotic tissues [peritoneal (n=19), ovarian (n=17) endometriosis and deep endometriotic nodules of the recto-vaginal septum (n=29)]. The relationship between aromatase expression and DLCs was traced by simple regression analysis. DLCs did not appear to be significant determinants of aromatase expression. CYP1A1 expression, measured as a positive control, was found associated with current smoking but not with DLCs. We conclude that DLCs do probably not facilitate the growth of endometriotic lesions by up-regulating the local expression of aromatase.     

Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro.

Many pesticides are able to block or activate the steroid hormone receptors and/or to affect the levels of sex hormones, thereby potentially affecting the development or expression of the male and female reproductive system or both. This emphasizes the relevance of screening pesticides for a wide range of hormone-mimicking effects. Twenty-two pesticides were tested for their ability to affect CYP19 aromatase activity in human placental microsomes using the classical [(3)H](2)O method. Prochloraz, imazalil, propioconazole, fenarimol, triadimenol, triadimefon (all fungicides), and dicofol (an acaricide) gave rise to a statistically significant inhibition of aromatase activity. The IC(50)s of prochloraz, imazalil, propioconazole fenarimol, triadimenol, and triadimefon were calculated from dose-response curves to be 0.04, 0.34, 6.5, 10, 21 and 32 microM, respectively. The IC(50) of dicofol was greater than 50 microM. The positive control 4-hydroxyandrostendione (1 microM) caused an inhibition of aromatase activity by 74%. The compounds, which did not affect the aromatase activity, were bromopropylate, chlorfenvinphos, chlorobenzilate, chlorpyrifos, diuron, heptachlor, iprodion, linuron, pentachlorphenol, procymidon, propyzamide, quintozen, tetrachlorvinphos and tetradifon. With the purpose of comparing the results for fenarimol obtained with the microsomal system with data from an intact cell system, an aromatase assay based on JEG-3 cells was established. 4-Hydroxyandrostendione (1 microM) inhibited the aromatase activity in JEG-3 cells by 94%. The IC(50) for fenarimol in this system was 2 microM, slightly lower than that observed in the microsomal system. For the first time, fenarimol has been demonstrated to inhibit aromatase activity in human tissues and, furthermore, propioconazole, triadimefon, and triadimenol were identified as weak aromatase inhibitors. In conclusion, seven out of 22 tested pesticides turned out to be weak to moderate aromatase inhibitors in vitro, indicating the relevance of elucidating the endocrine effects in vivo of these- compounds.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells.

Diindolylmethane (DIM) is an acid-catalyzed condensation product of indole-3-carbinol, a constituent of cruciferous vegetables, and is formed in the stomach. DIM alters estrogen metabolism and inhibits carcinogen-induced mammary tumor growth in rodents. DIM is a weak agonist for the aryl hydrocarbon (Ah) receptor and blocks the effects of estrogens via inhibitory Ah receptor-estrogen receptor cross-talk. DIM and various structural analogs were examined in H295R cells for effects on 3 cytochrome P450 (CYP) enzymes involved in estrogen synthesis and/or metabolism: CYP1A1, CYP1B1, and CYP19 (aromatase). Aromatase activity was measured by conversion of 1 beta-(3)H-androstenedione to estrone and (3)H(2)O. H295R cells were exposed to the test chemicals dissolved in dimethyl sulfoxide for 24 h prior to analyses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (0--30 nM) and DIM (0--10 microM) induced ethoxyresorufin-O-deethylase (EROD) activity, as a measure of CYP1A1 and possibly 1B1 activity, with EC(50) values of about 0.3 nM and 3 microM, respectively. DIM, but not TCDD, induced aromatase activity with an apparently maximal 2-fold increase at 10 microM; higher concentrations of DIM and many of its analogs were cytotoxic. TCDD (30 nM) significantly increased CYP1A1 and 1B1 mRNA levels, but had no effect on mRNA for CYP19. DIM (3 microM) significantly increased mRNA levels for all three CYPS: DIM analogs with substitutions on the 5 and 5' position (3 microM) induced aromatase and EROD activity, together with mRNA levels of CYP1A1, 1B1, and 19; analogs that were substituted on the central carbon of the methane group showed little or no inductive activity toward the CYPS: In conclusion, DIM and several of its analogs appear to induce CYPs via multiple yet distinct pathways in H295R human adrenocortical carcinoma cells.     

Synergistic stimulation of aldosterone production in human adrenocortical carcinoma NCI-H295R cells by endothelin-1 and angiotensin II

Aldosterone has recently been implicated in the pathogenesis of heart failure. The purpose of the present study was to determine the effect of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors that are also involved in heart failure, on aldosterone secretion by human adrenocortical carcinoma NCIH295R cells grown in 96-well plates. Ang II stimulated the production of aldosterone dose-dependently in serum-free medium, and the presence of serum drastically decreased aldosterone secretion. In contrast, ET-1-stimulated aldosterone production absolutely required serum. Under optimal conditions, ET-1 was more effective than Ang II as an aldosterone secretagogue. In a suboptimal condition of 2.5% serum, ET-1 and Ang II at 1 microM produced 63 and 76 pmol aldosterone/mg protein, respectively, while 230 pmol aldosterone/mg protein was generated upon coincubation with ET-1 and Ang II. The effect of ET-1 was inhibited dose-dependently by the selective ETA receptor antagonist BQ-123 with an IC50 of 23 nM, but the selective ETB receptor antagonist RES-701 had no effect up to 10 microM. These results suggest that ET-1 and Ang II stimulated aldosterone secretion synergistically in NCIH295R cells and that the effect of ET-1 was mediated via the ETA receptor subtype.     

PCB126 induces differential changes in androgen, cortisol, and aldosterone biosynthesis in human adrenocortical H295R cells.

Dioxins and polychlorinated biphenyls (PCBs) have been shown to accumulate in the adrenal glands when incorporated into the body. However, the impacts of exposure on adrenal steroidogenesis have not been thoroughly investigated. In this study, we demonstrated that dioxin-like PCB126 altered androgen, cortisol, and aldosterone biosynthesis differentially in human adrenocortical H295R cells. PCB126 diminished basal and cAMP-induced androstenedione production as well as CYP17 mRNA expression in a dose-dependent and time-dependent manner. The CYP17 repression was accompanied with decreases in the encoded 17 alpha-hydroxylase and 17,20-lyase activities, particularly the latter. In contrast, high concentrations of PCB126 stimulated basal cortisol and aldosterone biosynthesis, including induction of CYP21B, CYP11B1, and CYP11B2 mRNA expression and elevation of the conversion of cortisol from 17-OH-progesterone and aldosterone from progesterone. cAMP abolished the positive effect of PCB126 on cortisol synthesis, while it synergistically enhanced PCB126 stimulation on CYP11B2 expression and aldosterone production. It seemed likely that the downregulation of CYP21B caused by the combination of PCB126 and cAMP counteracted the CYP11B1 induction stimulated by the co-treatment. In addition, high concentrations of PCB126 might sensitize the regulation of adrenocorticotropin (ACTH) on the adrenocortical cells by increasing ACTH receptor levels. Because adrenal steroids have profound influences on glucose tolerance, insulin sensitivity, lipid metabolism, obesity, vascular function, and cardiac remodeling, this article also discusses the potential association of the detected adrenocortical alterations with increased diabetic and cardiovascular risk found among highly exposed people.     

Inhibitory effect of efonidipine on aldosterone synthesis and secretion in human adrenocarcinoma (H295R) cells

Targeting aldosterone synthesis and/or release represents a potentially useful approach to the prevention of cardiovascular disease. Aldosterone production is stimulated by angiotensin II (Ang II) or extracellular K+ and is mediated mainly by Ca2+ influx into adrenal glomerulosa cells through T-type calcium channels. We therefore examined the effects of efonidipine, a dual T-type/L-type Ca2+ channel blocker, on aldosterone secretion in the H295R human adrenocarcinoma cell line; 100 nmol/L Ang II and 10 mmol/L K+ respectively increased aldosterone secretion from H295R cells 12-fold and 9-fold over baseline. Efonidipine dose-dependently inhibited both Ang II- and K+-induced aldosterone secretion, and nifedipine, an L-type Ca2+ channel blocker, and mibefradil, a relatively selective T-type channel blocker, similarly inhibited Ang II- and K+-induced aldosterone secretion, but were much less potent than efonidipine. Efonidipine also lowered cortisol secretion most potently among these drugs. Notably, efonidipine and mibefradil also significantly suppressed Ang II- and K+-induced mRNA expression of 11-beta-hydroxylase and aldosterone synthase, which catalyze the final two steps in the aldosterone synthesis, whereas nifedipine reduced only K+-induced enzyme expression. These findings suggest that efonidipine acts via T-type Ca2+ channel blockade to significantly reduce aldosterone secretion, and that this effect is mediated, at least in part, by suppression of 11-beta-hydroxylase and aldosterone synthase expression.     

Induction of neutral endopeptidase (NEP) activity of SK-N-SH cells by natural compounds from green tea

Deposition of amyloid beta-peptide as senile plaques in the brain is one of the neuropathological hallmarks of Alzheimer's disease, which is the most prevalent progressive neurodegenerative disease leading to dementia. Neutral endopeptidase is one of the major beta-amyloid-degrading enzymes in the brain. To examine the influence of different polyphenols and other natural products from green tea extract (from Camellia sinensis, Theaceae), we used the neuroblastoma cell line SK-N-SH and studied the changes in the specific cellular neutral endopeptidase activity after long-term treatment with these substances. We have shown that caffeine leads to an increase in specific cellular neutral endopeptidase activity more than theophylline, theobromine or theanine. We have also shown that the combination of epicatechin, epigallocatechin and epigallocatechingallate with caffeine, theobromine or theophylline induced cellular neutral endopeptidase activity. It is suggested that the enhancement of cellular neutral endopeptidase activity by green tea extract and its natural products might be correlated with an elevated level of intracellular cyclic adenosine monophosphate.