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Inhibition of human placental aromatase activity by hydroxylated polybrominated diphenyl ethers (OH-PBDEs)
Rocío F. Cantón a,*, Deborah E.A. Scholten ª, Göran Marsh b, Paul C. de Jong ℃, Martin van den Berg ª
a Institute for Risk Assessment Sciences (IRAS), Utrecht University, PO Box 80176 3508 TD Utrecht, The Netherlands
b Department of Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden
St. Antonius Hospital, PO Box 2500, 3430 EM Nieuwegein, The Netherlands
Received 13 July 2007; revised 25 September 2007; accepted 27 September 2007 Available online 5 October 2007
Abstract
Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in many different polymers, resins and substrates. Due to their widespread production and use, their high binding affinity to particles, and their lipophilic properties, several PBDE congeners can bioaccumulate in the environment. As a result, PBDEs and their hydroxylated metabolites (OH-PBDEs) have been detected in humans and various wildlife samples, such as birds, seals, and whales. Furthermore, certain OH-PBDEs and their methoxylated derivatives (MeO-PBDEs) are natural products in the marine environment. Recently, our laboratory focused on the possible effects on steroidogenesis of PBDEs and OH-PBDEs, e.g. in the human adrenocortical carcinoma (H295R) cell line indicating that some OH-PBDEs can significantly influence steroidogenic enzymes like CYP19 (aromatase) and CYP17.
In the present study, human placental microsomes have been used to study the possible interaction of twenty two OH-PBDEs and MeO-PBDEs with aromatase, the enzyme that mediates the conversion of androgens into estrogens. All OH-PBDE derivates showed significant inhibition of placental aromatase activity with IC50 values in the low micromolar range, while the MeO-PBDEs did not have any effect on this enzyme activity. Enzyme kinetics studies indicated that two OH-PBDEs, 5-hydroxy-2,2’,4,4’-tetrabromodiphenyl ether (5-OH-BDE47) and 6-hydroxy-2,2’,4,4’- tetrabromodiphenyl ether (6-OH-BDE47), had a mixed-type inhibition of aromatase activity with apparent K;/K; of 7.68/0,02 µM and 5.01/ 0.04 µM respectively.
For comparison, some structurally related compounds, a dihydroxylated polybrominated biphenyl, which is a natural product (2,2’-dihyroxy- 3,3’,5,5’-tetrabromobiphenyl (2,2’-diOH-BB80)) and its non-bromo derivative were also included in the study. Again inhibition of aromatase activity could be measured, but their potency was significantly less than those observed for the OH-PBDEs.
These results show that a wide range of OH-PBDEs have the potential to disturb steroidogenesis and indicate a potential mechanism of action of these brominated flame retardant derivatives as endocrine disruptors in humans and wildlife. @ 2007 Elsevier Inc. All rights reserved.
Keywords: Flame retardants; Polybrominated diphenyl ethers (PBDEs); Metabolites; Aromatase activity; Human placenta
Introduction
Polybrominated diphenyl ethers (PBDEs) are chemicals used as flame retardants in all kinds of materials for electronic and daily apparatuses. PBDEs act in the gas phase of the fire by
reacting with the free radicals that are generated during the combustion process, thus terminating the reaction (Rahman et al., 2001).
PBDEs can be divided in three commercial mixtures, the pentaBDE, the octaBDE, and the decaBDE. Their global production was about 70,000 tons in 2001 (Hites, 2004) and approximately 50% is used in North America. The pentaBDE and octaBDE product mixtures have recently been banned in Europe and the production of those was stopped in North
* Corresponding author. Institute for Risk Assessment Sciences (IRAS), University of Utrecht, Yalelaan 2, 3508 TD, Utrecht, The Netherlands. E-mail address: r.Fernandezcanton@uu.nl (R.F. Cantón).
| OH-position | Structure | Aromatase inhibition (% of control at 40 µM) | Aromatase inhibition (IC50 µM) | |
|---|---|---|---|---|
| 1 Tri | 2'-OH-BDE28 Br OH O Br | 7.72±1.19 | 12.27 | |
| 2 Tri | Br 2'-MeO-BDE28 Br OMe O Br Br | n.e. | - | |
| 3 | Tri | 4'-OH-BDE17 Br Br O Br OH | 2.65±1.4 | 10.58 |
| 4 Tri | 4'-MeO-BDE17 Br Br | |||
| O | n.e. | - | ||
| 5 Tetra | Br OMe 2'-OH-BDE66 Br OH | |||
| O Br Br Br 2'-MeO-BDE66 | 0.13±0.04 | 5.22 | ||
| 6 | Tetra | Br OMe 0 Br Br Br 2'-OH-BDE 68 | n.e. | - |
| 7 | Tetra | Br OH 0 Br Br Br | 2.39±0.34 | 14.42 |
| Tetra | 2'-MeO-BDE 68 | n.e. | ||
| 8 | Br OMe 0 Br Br | - | ||
| Tetra | Br 3-OH-BDE47 | |||
| 9 | Br Br O OH | 27.49±3.25 | 20.74 | |
| Br Br 3-MeO-BDE47 | ||||
| 10 | Tetra Tetra | Br Br O OMe Br Br 4-OH-BDE42 | n.e. | - |
| Br Br | ||||
| 11 | O Br Br OH | 2.24±0.62 | 5.22 | |
| 12 Tetra 13 Tetra | 4-MeO-BDE42 Br Br O Br Br OMe | n.e. | - | |
| 4'-OH-BDE49 | ||||
| Br Br O Br OH Br 4'-MeO-BDE49 Br Br | 6.64±1.65 | 5.53 | ||
| 14 | Tetra | O Br Br OMe | n.e. | - |
| OH-position | Structure | Aromatase inhibition (% of control at 40 uM) | Aromatase inhibition (IC50 µM) |
|---|---|---|---|
| 15 Tetra | 5-OH-BDE47 Br Br O Br Br | 4.57±1.08 (a) | 8.72 |
| 16 Tetra | OH 5-MeO-BDE47 Br Br | ||
| 0 Br Br OMe 6-OH-BDE47 | n.e. | - | |
| Br ỌH | |||
| 17 Tetra 18 Tetra | 0 | 2.44±0.24 (a) | 7.44 |
| Br Br Br 6-MeO-BDE47 | |||
| Br OMe | |||
| O | n.e. | - | |
| Br Br Br 6'-OH-BDE49 | |||
| Br ỌH | |||
| 19 Tetra | O Br | 54.09±5.7 | 45.88 |
| Br Br 6'-MeO-BDE49 | |||
| Br OMe | |||
| 20 Tetra | O Br | n.e. | - |
| Br Br 6-OH-BDE90 | |||
| Br OH | |||
| 21 Penta | O Br | 5.22±1.39 | 6.51 |
| 22 Penta | Br Br Br 6-MeO-BDE90 Br OMe | ||
| O Br Br Br Br | n.e. | - |
Two independent experiments were done (independent human placenta); each concentration was tested in triplicate in each experiment and reproducible results were obtained. Graph shows one representative experiment. The results are presented as means with their standard deviations (SD). n.e., no effect. (a) mixed competitor.
America, though voluntarily action by industry. As a result of their widespread use, PBDEs are now commonly found in the abiotic and biotic environment, several of these compounds are lipophilic and environmental persistent. As a result of these properties PBDEs have found to bioaccumulate and biomagnify through the wildlife and human food chain (Gill et al., 2004; Hites, 2004; Birnbaum and Cohen Hubal, 2006). In humans, PBDE levels are generally higher in North America than other parts of the world and blood samples from the general U.S. population have been found to be in the range of 4-366 ppb with a median of 26 ppb. The lowest observed adverse effect level (LOAEL) value of the commercial pentaBDE mixture is considered to be 1 mg/kg/ day, which is based on the behavioral effects observed in mice
during a critical period after birth. Higher LOAEL values have been estimated for the octaBDE and decaBDE mixtures (Darnerud et al., 2001).
Several studies with rodent have shown that PBDEs can be metabolized to more polar compounds with a hydroxy, dihydroxy or hydroxy-methoxy structure (Hakk and Letcher, 2003; Malmberg et al., 2005; Marsh et al., 2006). It is known that several of these OH-PBDEs are retained in the blood in fish, bird, and mammalian species (Marsh et al., 2004; Malmberg et al., 2005; Verreault et al., 2005a) including man (Bergman et al., 2006). MeO-PBDEs have for example been detected in Baltic Sea salmon (Salmo salar) blood. However, the origin of MeO-PBDEs and OH-PBDEs from the marine environment is believed to be of natural origin (Marsh et al., 2004; Teuten et al., 2005). OH- and MeO-PBDEs are known natural products that have been found in marine algae, sponges and bacteria (Malmvarn et al., 2005). A number of these OH-and MeO- PBDEs have been isolated and structurally identified (Schuma- cher, 1995). All naturally occurring OH-PBDEs in these marine organisms have most exclusively hydroxy group in the ortho position in common and are exemplified by 6-OH-BDE47 and 2’-OH-BDE68. In the environment, MeO-PBDEs may be formed from OH-PBDEs via O-methylation in certain bacteria (Allard et al., 1987). It has also been suggested, but not proven, that hydroxylated metabolites of PBDEs may be formed by methylation in the liver or in intestinal microflora of vertebrates (Haglund et al., 1997; Asplund et al., 1999; Hakk and Letcher, 2003).
Several, mostly in vitro studies have shown that PBDEs can have potential endocrine disrupting properties from which a number of these can be attributed to the hydroxylated metabolites. OH-PBDEs can bind competitively to transthyretin (TTR), the thyroid hormone transport protein (Meerts et al., 2000), and cause estrogenic effects through interaction with the estrogen receptor (Meerts et al., 2001).
Previous studies from our laboratory have used the human adrenocortical carcinoma (H295R) cell line as in vitro system to assess effects of PBDE and some of their metabolites on steroi- dogenesis. These experiments show that especially OH-PBDEs could significantly inhibit CYP19 (aromatase) and CYP17 acti- vity, both key enzymes in steroidogenesis (Canton et al., 2005).
In humans, the placenta has a high expression of aromatase determining largely the fetal exposure to estrogens during this sensitive developmental stage (Simpson et al., 2001). Further- more, the placenta is also an essential tissue for blood flow between mother and the fetus, making it an effective way of transferring maternal PBDEs and/or their metabolites to the unborn.
In the present study, human placental microsomes were used to assess possible inhibitory effects of a series of OH- and MeO- PBDEs on aromatase. For comparison, some structurally related compounds i.e. dihydroxylated and dimethoxylated biphenyls were studied. Two of those, i.e. 2,2’-dihyroxy-3,3’,5,5’- tetrabromobiphenyl (2,2’-diOH-BB80) and 2,2’dimethoxy- 3,3’,5,5’-tetrabromobiphenyl (2,2’-diMeO-BB80), are present as natural products in marine environment (Isnansetyo and Kamei, 2003; Marsh et al., 2005).
Materials and methods
Chemicals. In this study, human placental microsomes were exposed to the following hydroxylated and methoxylated polybrominated diphenyl ethers (OH- PBDEs and MeO-PBDEs), which all were prepared as described elsewhere (Marsh, 2003) and abbreviated according to Ballschmiter and Buyten (1993): 2’-OH-BDE28, 2’-MeO-BDE28, 2’-OH-BDE66, 2’-MeO-BDE66, 2’-OH- BDE68, 2’-MeO-BDE68, 3-OH-BDE47, 3-MeO-BDE47, 4’-OH-BDE17, 4’- MeO-BDE17, 4-OH-BDE42, 4-MeO-BDE42, 4’-OH-BDE49, 4’-MeO-BDE49, 5-OH-BDE47, 5-MeO-BDE47, 6-OH-BDE47, 6-MeO-BDE47, 6’-OH-BDE49, 6’-MeO-BDE49, 6’-OH-BDE90, 6’-MeO-BDE90 (Table 1). The following biphenyls were also tested: 2,2’-dihydroxybiphenyl, 2,2’-dimethoxybiphenyl both purchased from Acros Organics (Geel, Belgium), and 2,2’-dihyroxy- 3,3’,5,5’-tetrabromobiphenyl (2,2’-diOH-BB80) and 2,2’dimethoxy-3,3’,5,5’- tetrabromobiphenyl (2,2’-diMeO-BB80) (Fig. 2) which were prepared according to Marsh et al. (Marsh et al. ES&T 39, 2005, 8684).
All compounds had a purity of >99% (except for 2,2’-dimethoxybiphenyl 98%) and the presence of brominated dibenzo-p-dioxins or dibenzofurans was eliminated by applying a charcoal column clean up as described earlier (Örn et al., 1996). Concentrations tested ranged from 0.04 µM up to 0.4 mM. This concentration range was selected based on earlier experiments in our laboratory with H295R cell line and a variety of different phytochemicals, pesticides and polybrominated diphenyl ethers (Sanderson et al., 2001).
Aromatase assay. The catalytic activity of aromatase was determined based on the tritiated water release method of Lephart and Simpson (1991). The method measures the production of 3H2O, which is formed as a result of the
Hydroxyl-BDEs
150
· 2-OH-BDE28
Aromatase Activity (pmol/min/mg prot)
· 2-OH-BDE66
· 2-OH-BDE68
100
HHHH
· 3-OH-BDE47
· 4-OH-BDE17
50
0
-6
5
-2
-1
0
1
2
3
log [ uM ]
Hydroxyl-BDEs
150
· 4-OH-BDE42
Aromatase Activity (pmol/min/mg prot)
a 4-OH-BDE49
. 5-OH-BDE47
100
· 6-OH-BDE49
· 6-OH-BDE47
· 6-OH-BDE90
50
0
-6-5
-2
-1
0
1
2
3
log [ [M ]
aromatization of the substrate [1B-3H] androstenedione. Human placental microsomes were exposed to 357 nM ([1B-3H] androstenedione) (New England Nuclear Research Products, Boston, MA, USA) dissolved in HEPES/MgCl2 buffer (50 mM/5 mM). Following the procedure described earlier (Lephart and Simpson, 1991), 200 ul of microsomal buffer was extracted and used for measuring the level of radioactivity after a substrate incubation period of 40 min. Corrections were made for background radioactivity, dilution factor, and specific activity of the substrate. Every experiment included human placenta microsomes exposed to 4-OH-Androstenedione, as a control positive of aro- matase inhibition, with an IC50 of 0.8 nM.
Human placenta microsome fraction. Two placentas were obtained from healthy pregnancies after delivery from the St. Antonius Hospital (Nieuwegein, The Netherlands) with informed consent of the patients (TME/Z-02-09, Medical Ethical Committee, St. Antonius Hospital, Nieuwegein, The Netherlands) and stored at -70 ℃. In order to isolate the microsomal fraction from the human tissue, the samples were weighed and homogenized in 10 volumes of Tris-HCI buffer (Tris-HCI 50 mM; 1.15% KCI) using a potter Elvehjem device. There after the tubes were centrifuged for 25 min at 15,000 rpm at 4 ℃. The supernatant was pipetted into a clean ultra-centrifuge tube and centrifuged for 1:15 h at 47,000 rpm at 4 ℃. Then, the supernatant was decanted and the pellet resuspended in sucrose solution (0.25 M). After that, 3 ul of suspension in tubes was taken with 147 ul milliQ for protein measurement and the microsome suspension was frozen in aliquots (50/100 ul) at -70 ℃ and stored until use.
Protein content was measured according to methods described earlier (Lowry et al., 1951). Protein levels were extrapolated from a standard curve that was generated using bovine serum albumin (Sigma A7030).
Enzyme kinetic assay. An enzyme inhibitor can be described as competitive or non-competitive. Competitive inhibition of enzyme activity occurs when a compound binds to the substrate-binding site of the enzyme while non- competitive inhibitors bind to allosteric sites of the enzyme. In the presence of a competitive inhibitor, Vmax can be reached if sufficient substrate is in solution, one-half Vmax needs a higher concentration of substrate compared to the control situation, and therefore Km is larger. With non-competitive inhibition, enzyme rate Vmax is reduced for all values of substrate, but Km is not affected because the active site of the enzyme molecules is unchanged. There are compounds which exhibit both types of inhibition, competitive and non-competitive, and in this mixed-type inhibitors, Vmax increases while Km decreases with higher concentrations.
In order to explain the mechanism of aromatase inhibition by OH-PBDEs, enzyme kinetic experiments were done using different concentrations of substrate (3H-androstenedione) (25-1000 nM) and different inhibitors (OH- BDEs) at 0.04 to 40 µM. Nonlinear regressions were done using Prism 3.0 (GraphPad Software Inc. San Diego, CA, USA) in order to calculate Km and Vmax values. Ki and K; values for mixed inhibitors were estimated from the linear parts of the slopes obtained by plotting respectively Km/Vmax and 1/V max, versus inhibitor concentrations.
Data analysis. All experiments were done in duplicate using two independent human placentas and results obtained with the OH-BDEs were not statistically different. Within an individual experiment each concentration was tested in triplicate. Human placentas were not pooled; therefore each graph shows one representative experiment. Graphs, statistical significant differences among means (one-way ANOVA), and IC50 calculations were done using Prism 3.0 (GraphPad Software Inc. San Diego, CA, USA).
Results
Inhibition of aromatase activity
Human placental microsomes were used to determine the inhibitory effects of derivatives of PBDEs on aromatase (CYP19) activity. Twenty two OH- and MeO-PBDEs were tested at concentrations ranging from 0.04 uM to 0.4 mM (Table 1).
Exposure to the OH-PBDE resulted in a concentration- dependent decrease of aromatase activity in all cases with IC50 values in the 5-10 µM range, except for 3-OH-BDE47 and 6- OH-BDE49 where IC50 values were higher than 20 µM (Fig. 1, Table 1). In general, the aromatase inhibitory potency of these
OMe
OH
O
O
OMe
OH
Biphenyl derivates
Aromatase Activity (% of control)
150-
· 2, 2’ OH-biphenyl
· 2, 2’ CH30-biphenyl
125-
I
100
75
50
*
25
0
-6
-5
-1
0
1
2
3
log [ uM ]
Br
OMe
Br
Br
O̧H
Br
Br
MeO
Br
Br
HO
Br
Biphenyl derivates
150
· 2,2’-OH-BB80
Aromatase Activity (% of control)
· 2,2’-CH30-BB80
£
100
50
0
-6
-5
-1
0
1
2
3
log [ uM ]
OH-PBDEs is at the same range and the low observed effect level (LOELs) for most of these metabolites is around 1 µM. No relationship could be found with the potency of the OH-PBDE as aromatase inhibitor and the position of the hydroxy group in the molecule.
Methoxy analogues of these OH-PBDEs were also tested and none of these MeO-PBDEs showed a significant effect on placental aromatase activity at concentrations up to 0.4 mM (data not shown).
Structurally related 2,2’-dihydroxybiphenyl and 2,2’- dimethoxybiphenyl and brominated biphenyl 2,2’-dihydroxy- 3,3’,5,5’-tetrabromo biphenyl (2,2’-diOH-BB80) and 2,2’- dimethoxy-3,3’,5,5’-tetrabromobiphenyl (2,2’-MeO-BB80) (Fig. 2) were used to better understand the inhibition of aromatase with respect to the position of the bromine atoms and the presence or absence of an ether bound in the molecule.
Results from exposed human placental microsomes showed a maximum of 50% (of control) inhibition of aromatase activity by 2,2’-dihydroxybiphenyl and 2,2’-diOH-BB80 at the highest concentration tested (0.4 mM).
Interestingly, inhibition of aromatase activity by 2,2’- dihydroxybiphenyl and 2,2’-diOH-BB80 was lower than in the
case of OH-PBDE metabolites exposed human placental microsomes, where aromatase activity was completely nullified, in the same concentration range.
As with the MeO-PBDEs, the methoxylated biphenyls (2,2’- diMeObiphenyl and 2,2’-diMeO-BB80) did not show any significant inhibitory effect on aromatase activity (Fig. 2).
Enzyme kinetics
In order to elucidate the mechanism of the aromatase in- hibition by these OH-PBDE compounds, human placental microsomes were exposed to different concentrations of two inhibitors, 5-OH-BDE47 and 6-OH-BDE47, in the range from 0.4 to 40 µM and substrate (3H-androstenedione) concentration from 20 nM up to 1 µM.
Nonlinear regression analysis of these experiments deter- mined apparent Km and Vmax values for aromatase activity (Figs. 3A and B). Vmax was decreased while Km increased when placental microsomes were exposed to higher concentrations of 5-OH- or 6-OH-BDE47. These inhibitors demonstrated mixed inhibition kinetics with corresponding Ki values of 7.68 and 5.01 µM and Ki values of 0.02 and 0.04 µM, respectively.
1200
Vmax [pmol/h/mg]
180
Km [nM]
160
1000
140
Vmax (pmol/h/mg)
800
120
100
Km (nM)
600
80
400
60
40
200
20
0
0
0
5
10
15
20
25
30
35
40
5 OH BDE47 (UM)
700
Vmax [pmol/h/mg]
70
Km [nM]
600
60
Vmax (pmol/h/mg)
500
50
400
40
Km (nM)
300
30
200
20
100
10
0
0
0
5
10
15
20
25
30
35
40
6 OH BDE47 (UM)
Discussion
Hydroxy PBDEs and aromatase inhibition
A concentration-dependent decrease of aromatase activity was found when human placental microsomes were exposed to different OH-PBDEs. Enzyme kinetic studies indicate that, in placental microsomes, OH-PBDEs showed a mixed-type in- hibition of aromatase activity with apparent Ki and Kį values of 7.68 µM and 0.02 µM in case of 5-OH-BDE47 and 5.01 µM and 0.04 µM for 6-OH-BDE47. The fact that Ki was several orders of magnitude higher indicates a more competitive than non- competitive mechanism of inhibition.
Comparison of apparent K¡ values of 5-OH-BDE47 and 6- OH-BDE47 indicates similar inhibitory potencies and mecha- nism of action. Thus, the position of the OH group, either ortho or meta, has no influence on the inhibition of aromatase. For comparison, fadrozole, a pharmaceutical aromatase inhibitor used in chemoprevention, has also been described as mixed-type aromatase inhibitor with apparent Ki value of 0.04 uM (Heneweer et al., 2004). However, the K; values of 5-OH- BDE47 and 6-OH-BDE47 indicate that these compounds are more than two orders of magnitude less potent aromatase inhibitors than fadrozole.
Previous experiments showed a decrease of aromatase activity in H295R cells after exposure to different OH-PBDE congeners. However, this was partly due to cytotoxicity (Canton et al., 2005). This is not considered likely in the case of placenta microsomes, indicating that aromatase inhibition is indeed caused by the metabolites of PBDEs.
Structure-activity relationships
The governing role in inhibition of steroidogenic enzymes such as CYP17 and aromatase (Canton et al., 2005, 2006) by OH- versus CH3O-groups in PBDE molecules is apparently not restricted to PBDEs only. This was also shown by Sanderson et al. (2004) who investigated the effects of various natural and synthetic flavonoids on the catalytic activity of aromatase in this H295R cell line. Some of these compounds (e.g. hydroxylated derivatives of flavonoids) were inhibitors of aromatase activity, whereas again their methoxylated analogues mostly lacked in- hibitory properties.
The presence of a hydroxyl group in these compounds ap- pears not only essential for aromatase inhibition, but also other potential endocrine disrupting effects. In vitro studies by Meerts and coworkers (Meerts et al., 2000, 2001) have shown that some of these OH-PBDEs bind competitively to the thyroid hormone transport protein transthyretin (TTR) and the estrogen receptor & (ER). Interestingly, less bromination adjacent to the hydroxy group or non bromination of the phenolic ring was necessary for a higher binding affinity with the ER&, while this decreased competitive binding to the human transthyretin (TTR).
These results are in agreement with those of Handayani and coworkers, who tested seven different hydroxy and methoxy- lated PBDEs for their antibacterial and fungicidal activities and in several cytotoxicity tests (Handayani et al., 1997). This study
showed that the absence of bromine relative to an ortho position of the hydroxyl group, a lower number of bromine atoms, and methylation of the hydroxy group on the phenolic ring, de- creased the cytotoxicity.
In the present study, no relationship was found between aromatase inhibition and position of the OH group. All hydroxy- PBDEs showed an IC50 in the lower micromolar range. In addition, some structurally related biphenyl derivatives were also included in our study. Up to 50% (of control) inhibition of aromatase activity could be measured by 2,2’-OH biphenyl and 2,2’-OH-PBB80 (Fig. 2), but similar to PBDEs, no effect was shown when the methoxylated analogue was used.
The results of our study show that the hydroxy group is not solely responsible for the inhibition of CYP19 activity; yet it is a combination of the hydroxy group, the adjacent bromine atoms, and the ether link that determine the effect. However, the role of the ether link appears the least important because aromatase activity was also inhibited, although at higher concentrations, by 2,2’-OH biphenyl as well as 2,2’-OH-PBB80, which both lack an ether link in the molecule.
Relevance for the human situation
These findings are an extension and in agreement with previous studies from our laboratory, which found that some OH-PBDEs can inhibit aromatase activity in the H295R human adrenocortical carcinoma cell line (Canton et al., 2005). In these earlier studies it was not always clear to which extent cytotoxicity contributed to the inhibition of aromatase. The use of placental microsomes eliminates the possibility of cytotoxicity as a confounding factor with aromatase inhibition by OH-PBDEs. Our present experiments with human placental microsomes indeed confirm the catalytic inhibition of aromatase by a wide range of OH-PBDE congeners.
If the in vitro medium concentrations in our study are compared with internal blood or serum concentrations found in humans and wildlife, it could be postulated that in vivo aromatase inhibition can only be expected at concentrations that are effective in our in vitro experiments. For this it is assumed that in vitro medium properties approach those of, e.g. human blood or serum. In our study aromatase inhibition occurs at concentrations higher than 1 µM, which is more than three orders of magnitude higher than those in humans and wildlife. So far, concentrations of OH-PBDEs in blood, adipose, and liver tissues, of a limited number of fish, bird, and mammalian species including humans was found to be in the picomolar and low nanomolar range (Marsh et al., 2004; Sinkkonen et al., 2004; Valters et al., 2005; Verreault et al., 2005a,b).
For comparison pharmaceuticals like letrozole or fadrozole used in the chemoprevention of estrogen dependent tumors inhibit aromatase activity up to 80-90% of the activity at low nanomolar. To cause a similar inhibition of aromatase in human placental microsomes, OH-PBDEs concentrations between 5 and 10 µM are needed. One of the highest concentrations found in wildlife so far (salmon plasma) was around 200 pg/g wet weight, which is approximately 0.4 nM. This is still more than three orders of magnitude lower than the effective
concentrations of OH-PBDEs in our present study. Hence, the lower potency of OH-PBDEs (>10-3 less) in combination with low concentrations in wildlife and humans makes aromatase inhibition at background exposure less likely.
Inhibition of aromatase can lead to disruption and imbalance between androgens and estrogens. Aromatase activity is a key factor in skeletal development and mineralization in both males and females. In postmenopausal women, aromatase activity is crucial for estrogen production within tissues and consequently for bone mass maintenance.
Conclusions
Human placental microsomes are a good in vitro model to detect catalytic inhibition of aromatase activity, due to its high activity of CYP19. Compared to in vitro cellular systems (e.g. H295R cells) cytotoxic effects are avoided and the experimental procedure is less time consuming than those needed for cell lines. However, transcriptional effects like induction or down- regulation cannot be detected with this in vitro model.
The results from our study clearly show that, with respect to possible endocrine disrupting effects mediated via aromatase, more attention should be given to the role of PBDE metabolites and not focus only on the parent compounds. The potential role of hydroxylated metabolites of PBDEs in aromatase inhibition is also supplementary to their effects on the estrogen receptor and thyroid hormone transporting protein, which indicates mul- tiple potential mechanisms of action for endocrine disruption of these brominated flame retardants.
Acknowledgments
The work described in this paper was fully supported by the FIRE European project with contract number QLRT-2001- 00596.
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