Induction and Inhibition of Aromatase (CYP19) Activity by Various Classes of Pesticides in H295R Human Adrenocortical Carcinoma Cells
J. Thomas Sanderson,1 Joke Boerma, Gideon W. A. Lansbergen, and Martin van den Berg Institute for Risk Assessment Sciences (IRAS), University of Utrecht, P.O. Box 80176, 3508 TD Utrecht, The Netherlands
Received December 27, 2001; accepted March 28, 2002
Induction and Inhibition of Aromatase (CYP19) Activity by Various Classes of Pesticides in H295R Human Adrenocortical Carcinoma Cells. Sanderson, J. T., Boerma, J., Lansbergen, G. W. A., and Van den Berg, M. (2002). Toxicol. Appl. Pharmacol. 182, 44-54.
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 mito- chondrial 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 (≥10 [M; 24-h exposure). p.p’-DDE had no effect on aromatase activity or cell viability at 1 and 10 uM. Various imidazole-like fungicides were aromatase inhibitors. Imazalil and prochloraz were potent mixed inhibitors (K;/Ki = 0.04/0.3 and 0.02/0.3 [M, respectively), whereas propi- conazole, difenoconazole, and penconazole were less potent com- petitive inhibitors (K; = 1.9, 4.5, and 4.7 [M, respectively). Fena- rimol, 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 [M) and atrazine (30 (M) in- creased 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. @ 2002 Elsevier Science (USA)
Key Words: H295R; pesticides; fungicides; herbicides; organotin; aromatase; endocrine disruption; induction; inhibition; cAMP; atrazine; imidazole; triazole; triazine.
There is increasing evidence that certain environmental con- taminants have the potential to disrupt endocrine processes, which may result in reproductive problems, certain cancers and other toxicities related to (sexual) differentiation, growth, and development. Research has focused mainly on interactions with sex hormone receptors, particularly the estrogen receptor. More recently, other mechanisms of interference with endo- crine functions have gained attention, including effects on enzymes involved in steroid hormone synthesis and metabo- lism. Particularly the cytochrome P450 (CYP) enzymes re- sponsible for the highly specific reactions in the steroid bio- synthetic pathway (Miller, 1988) are of interest as potential targets, given their key role in the formation of various highly potent endogenous steroid hormones.
Several classes of (relatively) persistent pesticides, such as organotin compounds, DDT and several metabolites, and a number of imidazole-like fungicides are suspected or have been shown to interfere with steroidogenesis. Particular atten- tion has been given to the enzyme aromatase (CYP19) which catalyzes the final, rate-limiting step in the conversion of androgens to estrogens. It has been postulated that organotin compounds may cause endocrine-disruptive effects such as “imposex” (penis development in females) in molluscs by inhibiting aromatase (CYP19) activity (Fent, 1996). Various imidazole-like fungicides are known to inhibit aromatase ac- tivity in human placental (Mason et al., 1987; Ayub and Levell, 1988; Vinggaard et al., 2000) and rainbow trout ovar- ian microsomes (Monod et al., 1993), the latter indicating their potential to block natural estrogen-mediated responses such as vitellogenin synthesis in female oviparous species during re- production. Recently, p.p’-DDE, which has antiandrogenic properties (Kelce and Wilson, 1997), has been reported to increase aromatase protein in rat liver (You et al., 2001). Support is increasing for the hypothesis that estrogenic or
1 To whom correspondence should be addressed. Fax: +31-30-253-5077. E-mail: t.sanderson@iras.uu.nl.
AP
antiandrogenic effects of xenobiotics may, in certain cases, be mediated by increases in aromatase activity, and reversely, antiestrogenic or androgenic effects by a decrease.
A useful bioassay to screen for interferences with steroido- genesis is the H295R human adrenocortical carcinoma cell line, which expresses numerous steroidogenic enzymes, in- cluding aromatase (Staels et al., 1993; Rainey et al., 1994; Sanderson et al., 2000). We used the H295R cell line to screen a number of pesticides known or suspected to interfere with steroid hormone function for potential effects on the catalytic activity and mRNA expression of aromatase. These included the organotin compounds dibutyl-, tributyl-, and triphenyltin chloride, DDT and several metabolites, and 13 imidazole-like fungicides, including the antiandrogenic pesticides p,p’-DDE and vinclozolin (Kelce et al., 1997; LeBlanc et al., 1997; Baatrup et al., 2001).
MATERIALS AND METHODS
Cell culture conditions. H295R cells were obtained from the American Type Culture Collection (ATCC CRL-2128) and grown in 1:1 (v/v) Dulbec- co’s modified Eagle medium/Ham’s F-12 nutrient mix (DMEM/F12) contain- ing 365 mg/ml L-glutamine and 15 mM Hepes (GibcoBRL 31300-038). The medium was supplemented with 10 mg/L insulin, 6.7 µg/L sodium selenite, and 5.5 mg/L transferrin (ITS-G, GibcoBRL 41400-045), 1.25 mg/L bovine serum albumin (Sigma A9647), 100 U/L penicilline/100, µg/L streptomycin (GibcoBRL 15140-114), and 2% steroid-free replacement serum Ultroser SF (Soprachem, France). For the aromatase experiments cells were treated as described previously (Sanderson et al., 2000). In brief, cells (about 1-2 × 105 cells/well) in 24-well culture plates containing 1 ml medium per well were exposed to various concentrations of the organotin compounds (a gift from Dr. R. Pieters, IRAS, Utrecht, The Netherlands), DTT, and metabolites, and imidazole-like pesticides (Riedel-deHaen, Germany) dissolved in 1 µal of dimethyl sulfoxide (DMSO; D4540, Sigma-Aldrich, U.S.A.). Negative control cells received 1 ul of DMSO. As positive control for aromatase inhibition, cells were exposed to 1 µM of the known aromatase inhibitor 4-hydroxyan- drostenedione in DMSO; as positive control for induction, 100 µM of 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) dissolved in medium containing 0.1% DMSO was used. Unexposed cells were included as further controls. All treatments were tested in quadruplicate, unless stated otherwise; each concentration-response experiment was performed three times. For the mRNA isolation experiments, cells in 6-well plates were exposed to 4 pl DMSO or the test chemicals in DMSO; a positive control (100 µM 8-Br- cAMP) was included on each plate. Each treatment was tested in triplicate; each experiment was performed three times. DMSO at 0.1% had no effect on CYP19 expression or catalytic activity relative to unexposed cells. Protein concentrations were determined by the fluorometric method of Udenfriend et al. (1972), using bovine serum albumin (A9647, Sigma-Aldrich) as standard. All exposures were for 24 h.
Isolation and amplification of RNA. RNA was isolated using the RNA Insta-Pure System (Eurogentec, Belgium) according to the enclosed instruc- tions and stored at -70℃. RT-PCRs were performed using the Access RT-PCR System (Promega, U.S.A.) with various modifications reported pre- viously (Sanderson et al., 2000). The purity of the RNA preparations was verified by denaturing agarose gel electrophoresis. The primer pair used for CYP19 mRNA amplification was 5’-TTA-TGA-GAG-CAT-GCG-GTA-CC- 3’; 5’-CTT-GCA-ATG-TCT-TCA-CGT-GG-3’, resulting in an amplification product of 314 bp. As reference, RT-PCR was performed on -actin mRNA using the primer pair 5’-AAA-CTA-CCT-TCA-ACT-CCA-TC-3’ and 5’- ATG-ATC-TTG-ATC-TTC-ATT-GT-3’, according to the instructions of the
Access RT-PCR kit, except using 2 mM MgSO4, an annealing temperature of 54℃ and 25 cycles. B-Actin mRNA was found not to be affected by any of the treatments (DMSO, pesticides, or 8Br-cAMP) and could be used reliably as a reference amplification response. Detailed information on PCR conditions, reproducibility, and ability of the method to be used semiquantitatively was published previously (Sanderson et al., 2000). Amplification products were detected using agarose gel electrophoresis and ethidium bromide staining. Intensity of the ethidium bromide stains were quantified using a FluorImager (Molecular Dynamics, U.S.A.).
Aromatase assay. The catalytic activity of aromatase was determined based on the method of Lephart and Simpson (1991) with minor modifications. Cells were exposed to 54 nM 1B-[3H]androstenedione (New England Nuclear Research Products, U.S.A.) dissolved in serum-free (Ultroser SF-free) culture medium and incubated for 1.5 h at 37°℃ in an atmosphere of 5% CO2 and 95% air. All further steps were as reported previously (Letcher et al., 1999; Sanderson et al., 2000). Aromatase activity was expressed in picomoles of androstenedione converted per hour per milligram cellular protein. The spec- ificity of the aromatase assay based on the release of tritiated water was verified by measuring the production of estrone (the aromatization product of androstenedione), using a 125I-labeled double-antibody radioimmunoassay kit (DSL-8700; Diagnostic Systems Inc, U.S.A.), and by using 4-hydroxyandro- stenedione, an irreversible inhibitor of the catalytic activity of aromatase, to block the formation of tritiated water (Brodie et al., 1977). Under our condi- tions unexposed or DMSO-exposed cells had a basal aromatase activity of 1.4 ± 0.2 pmol/h/mg protein.
EROD assay. Ethoxyresorufin-O-deethylation (EROD) activity, a mea- sure of the catalytic activity of the CYP1A subfamily of cytochrome P450, was determined in H295R cells using a modification of the method described by Burke and Mayer (1974). First, H295R cells in 24-well plates were preinduced for 24 h with 30 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), because basal EROD activity was barely detectable (0.5 + 0.5 pmol/h/mg protein; Sanderson et al., 2001b). The next day, the TCDD-containing medium was removed and replaced with fresh medium, and then the cells were exposed to the organotin compounds for a further 24 h. Then, the medium was removed from the cells and the cells were washed twice with warm (37℃) phosphate- buffered saline (PBS). Cells were then exposed to 0.5 ml Tris buffer (50 mM, pH 7.8) containing 0.9% (w/v) NaCl, 6.25 mM MgCl2, 5 L&M 7-ethoxyresoru- fin and 10 µM dicumerol. Resorufin formation was measured on a Cytofluor 2300 (Millipore, U.S.A.) using an excitation wavelength of 530 nm and emission wavelength of 590 nm. The formation of resorufin was followed over time and was linear for at least 60 min, at 37℃. Under these conditions EROD activity in preinduced control cells was 4.1 + 0.8 pmol/h/mg protein.
In a previous publication from our laboratory H295R cells were shown to have a very low basal EROD activity and expression of CYP1A1 and CYP1B1 mRNA which was inducible by TCDD (Sanderson et al., 2001b), demonstrat- ing that a functional aryl hydrocarbon receptor pathway is present in this cell line. Preliminary unpublished results indicated that the catalytic activity of CYP1A1 and 1B1 toward the 2- and 4-hydroxylation of estradiol, respectively, was very low in TCDD-induced H295R cells. In addition, 16@-hydroxylation (a CYP3A-type activity) was undetectable in this cell line. These observations together show that the contribution of CYP1A1 and 1B1, even after induction by TCDD, to the metabolism of androgens and/or estrogens in H295R cells is negligible and not of influence on the measurement of aromatase activity.
MTT reduction assay. Mitochondrial function, as an indicator of cytotox- icity, was assessed by measuring the capacity of H295R cells to reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan (Denizot and Lang, 1986). MTT is reduced to the blue-colored formazan by the mitochondrial enzyme succinate dehydrogenase, which is considered a reliable and sensitive measure of mitochondrial function. The cells in each well on the 24-well plate were incubated for 30 min, at 37℃ with 0.5 ml of MTT (1 mg/ml) dissolved in serum-free medium. Then, the MTT solution was re- moved, and the cells were washed twice with PBS. The formazan formed in the cells was extracted by adding 1 ml of isopropanol and incubating for 10 min at room temperature. The isopropanol was added directly to a plastic cuvette
for spectrophotometric analysis (Shimadzu UV-160A, Shimadzu Benelux, Belgium) at an absorbance wavelength of 560 nm. MTT reduction was linear with time for about 45 min and was not affected by DMSO treatment.
Neutral red uptake assay. Cell and organelle membrane function, as an indicator of cytotoxicity, was determined by measuring the capacity of H295R cells to actively take up neutral red (Borenfreund and Puerner, 1985). Cells in 24-well plates were incubated for 45 min at 37℃ with 0.5 ml neutral red (0.04 mg/ml) dissolved in serum-free medium. The reaction medium was removed from the cells, which were then washed twice with PBS. The neutral red was extracted from the cells by incubating for 2 min with 1.5 ml 1% acetic acid/50% ethanol in distilled water. The extraction procedure was repeated and the two fractions were pooled. The absorbance of neutral red was measured at 540 nm.
Cyclic AMP measurements. Intracellular cAMP concentrations were de- termined using a commercial enzyme-linked immunoassay kit (DE0450, R&D systems, UK) according to the instructions provided. Briefly, H295R cells were exposed to the test compounds in 12-well plates for up to 6 h, and then the medium was removed and the cells were washed with 50 mM Tris buffer containing 0.9% NaCl (PBS was not used because phosphate interferes with the immunoassay). The cells were lysed for 20 min in 300 ul of 0.1 N HCI; then the lysate was transferred to a plastic vial (Eppendorf), vortexed, and centrifuged at 12,000g for 10 min. The supernatant was diluted 1 in 5 with the assay buffer provided by the kit and underwent all other steps, including an acetylation step according to the instructions of the supplier. Each assay was accompanied by a cAMP standard curve.
Data analysis. All responses are presented as means with standard devi- ations (n = 3 or n = 4). Statistically significant differences from control groups were determined by a two-tailed t test using a correction for multiple comparisons and a significance level of 0.05. Nonlinear regression analyses of enzyme kinetic data were performed using Prism 3.00 (GraphPad Software, Inc, San Diego, CA) in order to calculate apparent Km and Vmax values. Ki values for competitive inhibitors were determined by plotting apparent Km values versus inhibitor concentration. Ki and K; values for mixed inhibitors were estimated from the linear parts of the slopes obtained by plotting Km/V max and 1/V max, respectively, versus inhibitor concentration. Estimates of log Kow values were calculated using KowWIN (Syracuse Research Corporation, Syr- acuse, NY).
RESULTS
Organotin Compounds
A 24-h incubation of H295R cells with the organotin com- pounds dibutyltin (DBT), tributyltin (TBT), and triphenyltin (TPT) chloride resulted in concentration-dependent decreases in aromatase and EROD activities (Fig. 1, top panels). EROD activity was included as an nonsteroidogenic CYP enzyme activity and independent measure to determine whether any interaction of the organotin compounds with aromatase activity was in fact specific in nature. TBT appeared to be the most potent, causing decreases in both CYP activities at concentra- tions above 30 nM, whereas DBT and TPT decreased the activities at concentrations of 100 and 300 nM, respectively. Examination of the cytotoxic properties of the organotin com- pounds indicated that TBT decreased mitochondrial MTT re- duction and neutral red uptake in H295R cells significantly at concentrations above 30 nM, whereas DBT and TPT did not decrease these measurements until concentrations above 100 nM were used (Fig. 1, bottom panels). The effect of the organotin compounds on total protein content in the wells was
also determined, demonstrating the first statistically significant decreases in protein content at concentrations of 100 nM for TBT, and above 200 nM for DBT and TPT. When exposure of H295R cells to the organotin compounds was limited to the duration of the aromatase (1.5 h) and EROD assays (1 h), to investigate their ability to cause direct inhibition of catalytic activity, no effects occurred. The only exception was a slight, yet statistically significant 20% decrease in EROD activity at the highest tested concentration of 1000 nM TBT (not shown). This concentration of TBT also resulted in significant de- creases in MTT reduction and neutral red uptake (about 20%) in H295R cells after the 1-h exposure. Under the same assay conditions, positive controls for direct catalytic inhibition of aromatase and EROD activity, 4-hydroxyandrostenedione and a-naphthoflavone, respectively, caused 54 and 34% inhibition of the respective activities at 10 nM, and greater than 80% inhibition at 100 nM.
DDT and Metabolites
Exposure of H295R cells to DDT and various metabolites for 24 h resulted in decreased aromatase activities for the compounds p,p-DDT, o,p-DDT, and o,p-DDE, whereas p,p- DDE had no effect at the concentrations tested (Fig. 2). The decreased aromatase activities only occurred at cytotoxic con- centrations of the compounds, which were determined by the cell viability measurements MTT reduction and neutral red uptake (not shown).
Imidazole-like Fungicides
Concentration-response experiments with 13 selected imida- zole-like fungicides (for structures see Table 1) demonstrated several effects on aromatase activity in H295R cells after a 24-h incubation (Fig. 3). These included decreases, increases, or a biphasic initial increase followed by a decrease in the catalytic activity of aromatase.
Inhibitors of aromatase activity. Several imidazole-like fungicides decreased the activity of aromatase concentration dependently in H295R cells with widely differing potencies (Fig. 3, top panel). To determine whether the observed de- creases in aromatase activity were a specific effect or second- ary to cytotoxicity, concentration-response experiments were performed to determine the effect of the compounds on cellular MTT reduction and neutral red uptake. In the case of hexacon- azole, tebuconazole, and fenarimol the decreases in aromatase activity occurred at concentrations close to decreases in the two measures of cell viability. In the case of imazalil, prochloraz, difenoconazole, penconazole, and propiconazole, aromatase activity was inhibited at concentrations well below those caus- ing the first signs of cytotoxicity. Therefore the latter five compounds were selected to determine their ability to inhibit aromatase activity directly in the presence of the substrate 1B-[3H]androstenedione for the duration of the catalytic assay. For each of the fungicidal inhibitors the enzyme kinetics were
120
120
T
Aromatase activity (% control)
100
100
I
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EROD activity (% control)
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*
80
80
*
*
*
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*
60
Aromatase
60
EROD
40
DBT
40
DBT
TBT
*
TBT
20
TPT
20
TPT
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control)
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TBT
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TBT
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TPT
20
TPT
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Organotin concentration (nM)
Organotin concentration (nM)
determined in the absence or presence of various concentra- tions of substrate and inhibitor. Nonlinear regression analysis of these experiments determined apparent Km and V max values for aromatase activity and K; values for the fungicides (Fig. 4). Propiconazole (Fig. 4, top panel), penconazole, and difenocon- azole demonstrated competitive inhibition kinetics and had Ki values of 1.9, 4.5, and 4.7 µM, respectively. The potent inhib- itors prochloraz (Fig. 4, bottom panel) and imazalil demon- strated mixed inhibition kinetics and had Kj values of 0.037 and 0.015 µM, and K; values of 0.33 and 0.37 uM, respec- tively.
Inducers of aromatase activity. Vinclozolin and several other fungicides increased the activity of aromatase concentra- tion dependently in H295R cells (Fig. 3, bottom panel). The compounds nuarimol and diclobutrazole produced a biphasic response with strongly reduced aromatase activities at a con- centration of 100 p.M. In the case of diclobutrazole this de- crease occurred concomitantly with decreases in MTT reduc-
300
Aromatase activity (% control)
250
1 uM
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4-HA
pp-DDT
op-DDT
op-DDE
pp-DDE
8Br-CAMP
| Estimated Log Kowª | Aromatase inhibition (IC50, AM) | Aromatase induction | Estimated Log Kowª | Aromatase inhibition (IC50, UM) | Aromatase induction | ||
|---|---|---|---|---|---|---|---|
| Imazalil (imidazole) CI CH3 O CI CH CH2 CH2 N 1 N | 4.10 | 0.15 | - | Difenoconazole (triazole) CI 0 0 CI H3C 0 ÇH2 N. N N | 5.20 | 4℃ | - |
| Prochloraz (imidazole) H2 H3C C CH2 CI CI H2 O N C N H2 0 CI N | 4.13 | 0.1b | - | Diclobutrazole (triazole) CI OH CH3 CI C H H2 CH3 N H CH3 N N | 4.01 | + | |
| Fenarimol (pyrimidine) OH N CI N CI | 3.62 | 80ª | +/- | Hexaconazole (triazole) CI OH CI C-C-C-CH3 CH2 H2 H2 H2 12 N. 11 N N | 3.66 | 55ª | - |
| Nuarimol (pyrimidine) OH N F 1 N CI | 3.17 | 100€ | + | Paclobutrazole (triazole) CI H OH CH3 3 C C CH3 H, N H N CH3 N 11 | 3.36 | +/- | |
| Atrazine (triazine) a N V CHỊCH_NH N NHCH(CH3)2 | 2.82 | ++ | Penconazole (triazole) CI CI H Ť C-C-CH3 CH H2 H2 2 N. N N " | 4.67 | 20€ | - | |
| Vinclozolin CI CI 0 N o H3C 0 H =CH2 | 3.03 | ++ | Propiconazole (triazole) CI CI CH2 O 3 H2 C 0 H2 CH2 N 1 N N | 4.13 | 5€ | - | |
| Tricyclazole (triazole) S N N N CH3 | 2.48 | + | Tebuconazole (triazole) OH CH3 CI C-C H2 H2 CH3 CHCH3 N. 1 N N // | 3.70 | 50ª | +/- |
ª Log Kow estimates were calculated using software by the Syracuse Research Corporation.
b Mixed inhibitor.
‘ Competitive inhibitor.
d Inhibition may be largely due to cytotoxicity: mechanism of inhibition unknown; IC50 is an estimate.
e Nuarimol exhibits a biphasic response (Fig. 4): mechanism of inhibition unknown.
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Aromatase activity (% control)
120
Imazalil
Prochloraz
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Difenoconazole
Penconazole
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Hexaconazole
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4-HA
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Pesticide concentration (uM)
tion and neutral red uptake. In the case of nuarimol, however, cytotoxicity was not apparent at 100 µM, suggesting that it is an inhibitor at higher concentrations. Atrazine, a triazine her- bicide previously shown to induce aromatase activity (Sander- son et al., 2000), was included as a positive control. To delineate the mechanism of aromatase induction by these com- pounds, their effect on CYP19 mRNA levels was examined. Vinclozolin increased CYP19 mRNA levels in H295R cells (Fig. 5), as was previously reported for atrazine (Sanderson et al., 2000). Diclobutrazole did not increase CYP19 mRNA levels significantly; nuarimol was not tested. In addition, these compounds were tested for their ability to increase intracellular cAMP levels. The aromatase inducers vinclozolin and atrazine increased cAMP levels time dependently in H295R cells (Fig. 6); diclobutrazole had no effect. IBMX and forskolin, known to increase intracellular cAMP levels via inhibition of phospho- diesterase activity and stimulation of adenylate cyclase activ- ity, respectively, were used as positive controls. Forskolin increased cAMP levels rapidly, a response which reached a plateau between 2 and 4 h with a maximum increase about
3-fold higher than control. IBMX was less efficacious and acted slower, increasing cAMP levels to a maximum of about 1.8-fold at 4 h (Fig. 6). Atrazine and vinclozolin produced a time-response course similar to that of IBMX, with an apparent postmaximal decline after 4 h that was statistically significant only for atrazine. As IBMX increased cAMP to higher levels than atrazine or vinclozolin, a concentration-response experi- ment was performed to determine whether this corresponded with a greater ability of IBMX to induce aromatase activity. The weaker phosphodiesterase inhibitor genistein was also included as positive control. IBMX induced aromatase activity 148, 239, 340, and 295%, at 10, 30, 100, and 300 µM, respectively. In the same experiment, 10 µM genistein and 30 M atrazine induced aromatase activity 223 and 227%, respec- tively. A comparison of the aromatase induction efficacy of the various compounds with their ability to increase cAMP levels resulted in a highly significant correlation (Fig. 7).
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Amplification response ratio of CYP19/beta-actin (% control ratio)
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Atrazine
Vinclozolin
300
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Pesticide concentration
DISCUSSION
Organotin Compounds
Organotin compounds are highly toxic chemicals and ubiq- uitous environmental contaminants due to their persistence and wide use in industry, agriculture, and antifouling paints. Or- ganotins have been linked to endocrine-disruptive effects such as imposex in molluscs, and inhibition of cytochrome P450 (CYP) activities, including aromatase (CYP19) in fish. How- ever, the frequent suggestion that aromatase inhibition is the mechanism linking organotin exposure to imposex has so far not been supported by scientific evidence. Historical concen- trations of tributyltin in contaminated surface waters such as
350
CAMP concentration (% of control)
300
250
-IBMX
- Atrazine
200
-O-Vinclozolin
150
Diclobutrazole
100
Forskolin
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0
0
2
4
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8
Time (h)
boat harbors have been found to range anywhere from 0.001 to as high as 7.2 µg/L (Fent, 1996), corresponding to concentra- tions of 3 pM to 22 nM. Concentrations in fish have been found to be as high as 2 mg/kg (wet weight), which would correspond to about 6 uM if we equal wet weight to that of water. Experimentally determined bioconcentration factors for TBT in various fish species range anywhere from 400 to 4000 (Fent, 1996). As current and recent TBT concentrations in contami- nated surface waters range from very low to less than 1 nM, high-end estimates of TBT concentrations in contaminated fish would be about 4 pM or about 1.5 µg TBT/kg wet weight. LC50 values in fish range anywhere from 1 to 100 uM, dependent on the experimental conditions and species sensi- tivity (Fent, 1996). In comparison, inhibitory effects on CYPs in vitro have generally been observed in the environmentally unrealistic high micromolar range (Fent and Stegeman, 1991; Fent et al., 1998), and similarly, a study in vivo in scup (Stenotomus chrysops) saw decreases in various immunoreac- tive CYP enzymes only at an intraperitoneal exposure to a remarkable high 16.3 mg TBT/kg fish wet weight (Fent and Stegeman, 1993). In the present study, detailed concentration- response experiments in H295R cells using DBT, TBT, and TPT chloride demonstrated that although the organotins de- creased the activities of both CYP1A and CYP19 in the upper nanomolar range, the decrease appeared to be entirely due to quantitatively similar decreases in measures of cell viability. In agreement with our findings, various measures of impaired cellular energy status and general health, such as decreased ATP production, loss of mitochondrial membrane integrity, and apoptosis, are known to be caused by TBT at concentra- tions ranging from 50 to 1000 nM (Fent, 1996). Furthermore, direct catalytic inhibition experiments with the organotin com- pounds, which required relatively short exposure regimes of 1 to 1.5 h, were not able to demonstrate inhibition of aromatase or EROD activity in the 1-1000 nM concentration range. Thus, it could not be concluded that the organotin compounds had
Aromatase activity (% control)
450
forskolin
400
r = 0.992
350
300
IBMX
250
atrazine
genist.
200
vinclozolin
diclobutrazole
150
100
100
150
200
250
300
350
CAMP levels (% control)
any selective inhibitory properties toward these CYP enzymes. Our negative findings with respect to aromatase inhibition are supported by a field study in gastropods (Bolinus brandaris) off the Mediterranean coast of Spain (Morcillo and Porte, 1999). A population highly polluted with organotin compounds (100% incidence of imposex in females) had severely de- creased estradiol levels compared to a relatively uncontami- nated population (37% imposex), although testosterone levels were not as strongly reduced. Despite this, aromatase activities were not different between the two populations. Furthermore, a recent report points out that the reductions in steroid levels occur in the later stages of imposex development and are more likely a consequence than a cause of imposex (Oberdorster, 2001). Instead, it is suggested that certain peptide hormones are more likely to play an important role in masculinization of molluscs. These studies combined with our laboratory results indicate that the development of imposex and the action of organotin compounds occur via mechanism(s) other than inhi- bition of aromatase activity.
Imidazole-like Fungicides and Aromatase Inhibition
Various imidazole-like fungicides owe their fungicidal ac- tion to the specific inhibition of ergosterol biosynthesis in yeasts and fungi by inhibiting the CYP enzyme 14a-lanesterol demethylase. The selectivity of these fungicides is variable and some compounds are known to interact with several human CYP enzymes, including the steroidogenic CYP enzyme aro- matase (Mason et al., 1987; Ayub and Levell, 1988; Vinggaard et al., 2000). In the present study, we demonstrated the ability of 5 of the 13 tested fungicides to inhibit the catalytic activity of aromatase in vitro in the H295R human adrenocortical carcinoma cell line without causing cytotoxicity. In agreement with previous experiments in JEG-3 human placental chorio- carcinoma cells (Vinggaard et al., 2000) and human placental microsomes (Vinggaard et al., 2000), we found that imazalil and prochloraz were potent catalytic inhibitors of human aro- matase activity. We did not observe aromatase inhibition by fenarimol until cytotoxic concentrations (100 µM), which is in contrast to the in vitro inhibition reported previously (Hirsch et al., 1987; Vinggaard et al., 2000). Although Vingaard and co-workers report IC50 values for fenarimol of 2 uM in JEG-3 cells and 10 pM in placental microsomes, a closer examination of their results suggests IC50 values that are somewhat higher, at about 10 µM in JEG-3 cells and between 10 and 50 M in placental microsomes. Hirsch and co-workers measured an IC50 value of fenarimol for inhibition of rat ovarian microso- mal aromatase activity of 4.1 p.M. It is possible that aromatase inhibition in H295R cells is masked by the relative sensitivity of these cells to the cytotoxic effects of fenarimol. The aro- matase inhibitors propiconazole, penconazole, and difenocon- azole were competitive in nature, whereas the potent inhibitors imazalil and prochloraz demonstrated mixed inhibition kinet- ics, indicating that various mechanisms of enzyme inhibition
play a role. The mixed inhibition by imazalil and prochloraz is in contrast to their suggested competitive nature in other stud- ies (Mason et al., 1987; Monod et al., 1993). However, those studies made no attempt to perform enzyme kinetic studies using various concentrations of substrate and inhibitor to sup- port their claim. We point out that noncompetitive or mixed enzyme inhibition does not necessarily imply irreversibility, and that reversible inhibition does not imply competitiveness. Prochloraz and imazalil are structurally similar to various imidazole-containing drugs used clinically, such as the potent aromatase inhibitor fadrozole (Ki in lower nanomolar range) and numerous antifungal drugs shown to reversibly (although not necessarily competitively) inhibit aromatase activity in human placental microsomes (Ayub and Levell, 1988). To our knowledge, this is the first report to examine the nature of inhibition kinetics of these environmentally used fungicides toward human aromatase in a cellular system.
Imidazole-like Fungicides and Aromatase Induction
Five of the 13 tested fungicides increased aromatase activity. Vinclozolin appeared to be the most efficacious inducer, fol- lowed by diclobutrazole. Aromatase induction in H295R cells has been observed previously with the herbicide atrazine and several of its analogues and metabolites (Sanderson et al., 2000, 2001a). In these studies, atrazine induced aromatase activity about 2.5-fold and increased CYP19 mRNA levels between 1.5- and 2-fold. In the present study, beside atrazine only vinclozolin increased CYP19 mRNA levels in a statisti- cally significant manner.
It is possible that vinclozolin may exert part of its antian- drogenicity (Kelce et al., 1997; Baatrup et al., 2001) via aromatase induction if this mechanism were to occur in vivo. This may be the case for p,p’-DDE, which appeared to increase hepatic aromatase activity in rats in vivo, although not in rat hepatocytes in primary culture (You et al., 2001). However, this study failed to address an important confounding factor in the use of the tritiated water release assay to measure aroma- tization of 18-[3H]androstenedione in rat liver, namely the release of 3H2O by hydroxylation of 18-[3H]androstenedione at the 1ß position. This reaction is catalyzed partly by CYP3A1 and possibly CYP2B1 (Waxman et al., 1985), enzymes that are highly induced by p,p’-DDE in rat liver (You et al., 1999). Thus, it is crucial to validate the tritiated water release assay by measuring the aromatization product estrone and through in- hibition of the release by selective aromatase inhibitors such as 4-hydroxyandrostenedione or fadrozole. In any case, p,p’-DDE did not affect aromatase activity in H295R cells in our present study.
In H295R cells, steroidogenic CYPs are induced by syn- thetic cAMP analogues or stimulation of adenylate cyclase by forskolin, which results in increased intracellular cAMP levels. This, in turn, stimulates the cAMP-mediated protein kinase A pathway leading to increased gene transcription (Rainey et al.,
1993; Staels et al., 1993). Novel, yet consistent with this mechanism, is our finding that aromatase activity can also be induced by phosphodiesterase inhibitors, such as IBMX and genistein, which indirectly lead to increased levels of cAMP by inhibiting its degradation. Comparison of the time-response and dose-response curves for aromatase induction and cAMP induction by IBMX and the pesticides vinclozolin and atrazine show a good correlation. This suggests that the mechanism of aromatase induction by certain pesticides such as atrazine and vinclozolin may involve the inhibition of phosphodiesterase activity. This implies that these compounds would target cells/ tissues in which the expression of aromatase is regulated by cAMP-dependent promoters, including adrenal cortex, gonads, and (malignant) breast adipose (Simpson et al., 1993; Bulun and Simpson, 1994). It is noteworthy that aromatase expression in normal female breast adipose tissue is mainly regulated by the glucocorticoid-responsive promoter 1.4, with a minor con- tribution from the cAMP-responsive promoters pII and 1.3. In breast tissue from cancer patients, however, aromatase expres- sion is elevated and regulated mostly by the cAMP-responsive promoters (Agarwal et al., 1996; Zhou et al., 1997; Chen et al., 1999, 2001). These findings suggest that subpopulations with an increased risk of breast cancer due to the presence of cells that have switched promoter activity from glucocorticoid to cAMP responsiveness may be more susceptible to the effects of aromatase inducers that act by elevating cAMP levels, such as atrazine and vinclozolin.
Structure-Activity Relationships
It is difficult to delineate a relationship between the structure of the fungicides and their biological activity as inhibitors or inducers of aromatase. The most potent aromatase inhibitors in H295R cells were imazalil and prochloraz (K;/K; values in the upper nanomolar range), which both contain an imidazole and chlorinated aromatic moiety. The structurally similar imidazole antifungal drugs (in order of decreasing potency) tioconazole, econazole, bifonazole, clotrimazole, miconazole, and isocon- azole have previously been reported to be good inhibitors with similar potencies in various in vitro systems (Mason et al., 1987; Ayub and Levell, 1988). A group of somewhat less potent inhibitors in H295R cells was the triazole-containing fungicides difenoconazole, penconazole, and propiconazole (Ki values in the lower micromolar range). The structurally related triazole fungicides triademenol and triadimefon were also reported to have in vitro inhibition potencies of a similar magnitude (Vinggaard et al., 2000). On the other hand, two clinically used triazole-containing “designer” aromatase inhib- itors, letrozole and anastrozole, were found to be considerably more potent (K¡ values in the lower nanomalor range) (Santen and Harvey, 1999; Brodie, 2002). These compounds contain one or more carbonitrile groups, which contribute favorable to their potency and selectivity toward aromatase; these moieties are not present in our selected fungicides. The hydroxy-con-
taining triazoles tebuconazole, hexaconazole, and paclobutra- zole did not inhibit aromatase at subtoxic concentrations in H295R cells, as was true for the hydroxylated pyrimidine fungicides fenarimol (a known microsomal aromatase inhibi- tor) and nuarimol, possibly due to the relative sensitivity of the H295R cells to their toxicity. In the case of the hydroxylated triazoles there was a tendency to increase aromatase at subtoxic concentrations, which was most pronounced and statistically significant only for diclobutrazole.
Enzyme kinetic analyses indicated that the imidazole fungi- cides were mixed inhibitors, whereas the triazole fungicides were competitive inhibitors of aromatase with Ki values in the low micromolar range. It is unclear from the literature, how- ever, whether this is a general rule; the imidazole-containing aromatase inhibitor fadrozole has been reported to have com- petitive (Moslemi and Seralini, 1997) and noncompetitive (Yue and Brodie, 1997) properties. Consistent with the com- petitive nature of the triazole fungicides, letrozole, notwith- standing its far greater potency, was also found be a compet- itive inhibitor of microsomal aromatase (K; = 1.2 nM) in guinea pig brain (Choate and Resko, 1996). A comparison of the logarithm of the estimated octanol-water partitioning coef- ficients (log Kow) of the five nonhydroxylated triazole fungi- cides in the present study with the inverse of their respective IC50 values for inhibition of aromatase activity in H295R cells (Table 1) revealed a highly significant positive correlation (r = 0.951; P < 0.01; n = 5). This suggests that an important factor in the potency of the competitive triazole-containing inhibitors is their hydrophobicity, which likely reflects their combined ability to cross cellular membranes and to interact with the substrate pocket of the catalytic site of CYP19. The mixed inhibitors prochloraz and imazalil had inhibition poten- cies at least an order of magnitude greater than would be expected from their log Kow value (Table 1), again emphasizing that they act via (a) different mechanism(s) of inhibition from the triazoles.
The triazine-containing herbicides atrazine, simazine, and propazine and the fungicide vinclozolin were inducers of aro- matase activity, but did not appear to interact with the catalytic function of the enzyme directly. Atrazine and vinclozolin acted by increasing intracellular cAMP levels, possibly through in- hibition of phosphodiesterase activity, suggesting this enzyme as a target for SAR analysis of aromatase inducers in H295R cells. Various designer triazine-containing compounds are po- tent inhibitors of phosphodiesterase 4 in smooth muscle tissue (Leroux et al., 1999).
The complexity of potential effects of chemicals on aro- matase activity in various systems in vitro is illustrated by the isoflavone genistein. Genistein appears to be a weak aromatase inhibitor in Chinese hamster ovary (CHO) cells (Ki = 123 µM) (Kao et al., 1998) and rainbow trout ovarian microsomes (Pelissero et al., 1996), but is inactive in human placental microsomes (Pelissero et al., 1996; White et al., 1999). In human cell systems genistein has either no effect or increases
aromatase activity. The latter has been demonstrated in the present study using H295R cells and previously in HCT8, but not HCT116 human colon adenocarcinoma cells (Fiorelli et al., 1999). Given the complex biological activities of genistein and other natural flavonoids, some of which can act as more or less potent phosphodiesterase inhibitors (Kuppusamy and Das, 1992), tyrosine kinase inhibitors (Akiyama et al., 1987), weak (partial) estrogen agonists/antagonists (Martin et al., 1978; Collins et al., 1997; Le Bail et al., 1998), and weak aromatase inhibitors (Kao et al., 1998), various effects on aromatase activity can be expected, which will depend on the specific mechanism of regulation of the enzyme in the chosen in vitro system. It is possible that the differential effects of genistein in the two HCT human colon cell lines are due to differential cAMP responsiveness of aromatase expression in those lines. It is apparent that the fungicides in the present study also have various biological targets in H295R cells. The biphasic re- sponses observed occasionally in Fig. 3 may be explained if the test compound inhibits phosphodiesterase activity (or other- wise increases cAMP levels) at lower and inhibits aromatase activity at higher concentrations. Future experiments are un- derway to develop predictive structure-activity relationships for induction and/or inhibition of aromatase activity by envi- ronmental contaminants in various cell types.
CONCLUSIONS
We conclude that the inhibitory effects of the organotin compounds on aromatase and EROD activity in H295R human adrenocortical carcinoma cells occur at cytotoxic concentra- tions, indicating that the decreased CYP activities are not due to selective catalytic inhibition, but likely to the general and high affinity of organotins for sulfhydryl-containing proteins and/or secondary to decreased cell function. It was further found that various imidazole-like fungicides were capable of inhibiting aromatase activity through competitive (triazoles) or noncompetitive/mixed (imidazoles) mechanisms, whereas vin- clozolin and the triazine herbicide atrazine were inducers of aromatase transcription and catalytic activity by increasing intracellular cAMP levels, possibly through inhibition of phos- phodiesterase activity.
ACKNOWLEDGMENTS
We thank Dr. Edwin D. Lephart at Brigham Young University, Utah, for his advice on using the tritiated water release assay to measure aromatase activity. We are grateful to Bas Defize of the Hubrecht Laboratory at the University of Utrecht, The Netherlands, for the use of the FluorImager.
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