ELSEVIER

In vitro effects of brominated flame retardants and metabolites on CYP17 catalytic activity: A novel mechanism of action?

Rocío F. Cantón ª,*, J. Thomas Sanderson b, Sandra Nijmeijer ª, Åke Bergman d, Robert J. Letcher℃, Martin van den Berg ª

a Institute for Risk Assessment Sciences (IRAS), University of Utrecht, Yalelaan 2, 3508 TD, Utrecht, The Netherlands

b Institut National de la Recherche Scientifique, Institut Armand-Frappier (INRS-IAF), Université du Québec, Montréal, Québec, Canada H9R 1G6 ” National Wildlife Research Centre, Canadian Wildlife Service, Environment Canada, Carleton University, Ottawa, Ontario, KI A OH3, Canada d Department of Environmental Chemistry and Analytical Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden

Received 8 April 2006; revised 11 May 2006; accepted 12 May 2006 Available online 19 May 2006

Abstract

Fire incidents have decreased significantly over the last 20 years due, in part, to regulations requiring addition of flame retardants (FRs) to consumer products. Five major classes of brominated flame retardants (BFRs) are hexabromocyclododecane isomers (HBCDs), tetrabromobi- sphenol-A (TBBPA) and three commercial mixtures of penta-, octa- and deca-polybrominated diphenyl ether (PBDE) congeners, which are used extensively as commercial FR additives. Furthermore, concentrations of PBDEs have been rapidly increasing during the 1999s in human breast milk and a number of endocrine effects have been reported. We used the H295R human adrenocortical carcinoma cell line to assess possible effects of some of these BFRs (PBDEs and several of their hydroxylated (OH) and methoxylated (CH3O) metabolites or analogues), TBBPA and brominated phenols (BPs) on the combined 17a-hydroxylase and 17,20-lyase activities of CYP17. CYP17 enzyme catalyzes an important step in sex steroidogenesis and is responsible for the biosynthesis of dehydroepiandrosterone (DHEA) and androstenedione in the adrenals. In order to study possible interactions with BFRs, a novel enzymatic method was developed. The precursor substrate of CYP17, pregnenolone, was added to control and exposed H295R cells, and enzymatic production of DHEA was measured using a radioimmunoassay. In order to avoid pregnenolone metabolism via different pathways, specific chemical inhibitor compounds were used. None of the parent/precursor BFRs had a significant effect (P< 0.05) on CYP17 activity except for BDE-183, which showed significant inhibition of CYP17 activity at the highest concentration tested (10 µM), with no signs of cytotoxicity as measured by mitochondrial toxicity tests (MTT). A strong inhibition of CYP17 activity was found for 6-OH- 2,2’,4,4’-tetrabromoDE (6-OH-BDE47) with a concentration-dependent decrease of almost 90% at 10 µM, but with a concurrent decrease in cell viability at the higher concentrations. Replacement of the 6-OH group by a 6-CH3O group eliminated this cytotoxic effect, but CYP17 activity measured as DHEA production was still significantly inhibited. Other OH- or CH3O-PBDE analogues were used to elucidate possible structural properties behind this CYP17 inhibition and associated cytotoxicity, but no distinct structure activity relationship could be determined.

These in vitro results indicate that OH and CH3O-PBDEs have potential to interfere with CYP17 activity for which the in vivo relevance still has to be adequately determined.

Keywords: Brominated flame retardants (BFRs); Polybrominated diphenyl ethers (PBDEs); Hydroxylated PBDEs (OH-PBDEs); Methoxylated PBDEs (CH3O- PBDEs); CYP17; Dehydroepiandrosterone (DHEA); H295R human adrenocortical carcinoma cells

Introduction

Brominated flame retardants (BFRs) are used, among others, in plastics, electronic equipment, television sets, mobile

* Corresponding author. E-mail address: r.Fernandezcanton@iras.uu.nl (R.F. Cantón).

devises, building materials and textiles to increase their resistance to fire. BFRs have become an increasingly important group of organohalogen compounds, which include hexabro- mocyclododecane isomers (HBCDs), tetrabromobisphenol-A (TBBPA) and commercial mixtures of penta-, octa- and deca- brominated diphenyl ether (PBDE) congeners, which are extensively used at high production-volume levels (e.g., in

2001, the total market demand for PBDEs was 67,000 tonnes including 7500 tonnes of the penta-BDE product; Kierkegaard et al., 2004). Nowadays, penta- and octa-BDE mixtures have been banned in Europe and production voluntarily stopped in United States.

Some BFR compounds were found in environmental samples as long as 25 years ago, but they are now found globally in biota and accumulate in lipid-rich organisms, e.g., fatty fish and marine mammals (Law et al., 2003; Hites, 2004). Their physical and chemical properties can explain these increased levels in biotic tissues. Many BFRs are lipophilic and slowly metabolized; they also have high binding affinity to organic matter and a tendency to accumulate in sediment (Darnerud et al., 2001; Alaee et al., 2003).

In vitro studies have shown that certain BFRs can affect thyroid hormone homeostasis by acting as potent competitors of T4 for binding to human transthyretin and thyroid hormone receptors (Meerts et al., 2000; Zhou et al., 2002). Some PBDEs have also shown interactions with the estrogen receptor in vitro by stimulating an ER-mediated luciferase reporter gene (Meerts et al., 2001; Olsen et al., 2002). In addition, European concentrations of PBDEs in human milk have increased during the period 1972-1997, from 0.07 to 4.02 ng/g lipid weight, indicating a doubling in levels every 5 years (Noren and Meironyte, 2000). Recent studies have shown that PBDE levels seem to have reached a maximum value and are starting to decrease slowly (Sellstrom et al., 2003). This is probably related to the European ban on some BFR compounds, e.g., the penta-BDE mixture, during the last years. However, in North America levels are on average 10- 20 times higher compared to the European situation.

In exposed organisms, including humans, toxic effects, bioaccumulation, metabolism and pharmacokinetics are important criteria for the risk assessment of BFRs. In rats and mice, tissue distribution and metabolism of several PBDEs (e.g., 2,2’,4,4’-tetrabromoDE (BDE-47), 2,2’,4,4’,5- pentabromoDE (BDE-99) and 2,2’,3,3’,4,4’,5,5’,6,6’-decabro- moDE (BDE-209)) and TBBPA have been investigated (Orn and Klasson-Wehler, 1998; Hakk et al., 2002; Darnerud, 2003; Morck et al., 2003; Sandholm et al., 2003; Staskal et al., 2005; Sanders et al., 2006). BDE-47 and -99 have been shown to be rapidly absorbed and distributed among lipid rich tissues. In contrast, TBBPA and BDE-209 were rapidly excreted; resulting from a relatively fast metabolism and/or elimination (Sandholm et al., 2003; Thomas et al., 2005; Thuresson et al., 2005). In the case of BDE-209, biotransformation into hydroxyl (OH-) and methoxylated (CH3O-) PBDEs or lower brominated biphenyl ethers in combination with a low bioavailability in general from the gastrointestinal tract has been reported in dietary exposure studies with common carp (Stapleton et al., 2004).

OH- and CH3O-BDEs have also been reported in the blood and liver of wildlife and humans (Haglund et al., 1997; Asplund et al., 1999; Hovander et al., 2002; Hakk and Letcher, 2003; Valters et al., 2004). However, the origin of these derivatives remains controversial. Some of these

OH-PBDEs (e.g., 6-OH-BDE47) can be produced by marine organisms such as sponges or ascidians, but in higher vertebrate species P450 enzyme-mediated processes can also produce these OH-PBDEs. Moreover, HBCD isomers, 2,4,6- tribromophenol (TBP) and TBBPA, have found to be toxic to aquatic organisms and may have long-term adverse effects in the aquatic environment (Gribble, 1996; de Wit, 2002; Ronisz et al., 2004).

Consequently, the concern about BFRs and their deriva- tives with respect to their potential as endocrine disruptors has been growing in humans and wildlife during the last decade. Potential endocrine disruptor compounds may cause reproductive problems, certain cancers and other toxicities related to (sexual) differentiation, growth and development if present at sufficiently high levels. Several cytochrome P450 (CYP) enzymes are responsible for the highly specific reactions in the steroid biosynthesis pathway and are potential targets for endocrine disruption. These steroidogenic enzymes are responsible for the biosynthesis of various steroid hormones, including glucocorticoids, mineralocorticoids, pro- gestins and sex hormones (estrogens and androgens). Androgens and subsequently estrogens are ultimately derived from cholesterol via the formation of pregnenolone, 17-alpha- hydroxypregnenolone and DHEA, the latter two synthesized by CYP17. Androgens may subsequently be converted to estrogens by the enzyme aromatase (CYP19). Thus, both CYP17 and CYP19 catalyze key steps in the production of sex hormones in humans.

The CYP17 enzyme catalyzes two different enzymatic steps, steroid 17a-hydroxylase and 17,20-lyase activities, and is responsible for the production of DHEA, which is synthesized abundantly in the adrenal gland in humans (Chen and Parker, 2004). The plasma levels of DHEA rise continually from the age of 6 to 7, reaching a maximum in the second decade of life, after which it declines to about 15% of the peak level in the ninth decade of life.

Both in vitro and in vivo experimental studies strongly indicate that DHEA is related to anti-obesity, anti-tumor, anti-aging and anti-cancer effects (Ciolino et al., 2003). DHEA is a potent non-competitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH) and as a consequence lowers NADPH levels and reduces NADPH- dependent oxygen-free radical production. Furthermore, in rats, DHEA inhibited the expression of some hepatic carcinogen-activating enzymes like CYP1A1 and CYP1A2 (Labrie et al., 2003; Schwartz and Pashko, 2004).

Recent studies in our laboratory focused on potential interactions of a wide range of BFRs with sex hormone synthesis and metabolism. Previous results from our research group showed inhibitory and inductive effects by certain BFRs in the H295R human adrenocortical carcinoma cell line on aromatase (CYP19) activity. H295R cells express a large number of steroidogenic enzymes (Gazdar et al., 1990) and were also used in the present study, to develop a new enzymatic method for CYP17 activity measurement and to assess possible effects of selected BFRs and their metabolites.

Materials and methods

Brominated flame retardants and analogues. H295R cells were exposed to a selection of BFRs, i.e., tetrabromobisphenol-A (TBBPA), tetrabromobisphenol A-bis (2,3-dibromopropylether (TBBPA-DBPE or FR-720)) and 2,4,6-tribro- mophenol (TBP). A range of polybrominated diphenyl ethers (PBDEs) and their OH- or CH3O-containing derivatives (Tables 1 and 2) were synthesized as described elsewhere (Örn et al., 1996; Marsh et al., 1999). All BFRs were highly purified (> 99% purity) and the presence of brominated dibenzo-p-dioxins or dibenzofurans was eliminated by applying a charcoal column clean-up step (Örn et al., 1996). Stock solutions of 2.5 mM were used for further dilution to experimental concentrations ranging from 0.01 µM to 10 µM.

Cell culture and treatment. H295R cells were obtained from the American Type Culture Collection (ATCC CRL-2128) and grown in culture under conditions published previously (Sanderson et al., 2000). Briefly, the cells were grown in 1:1 Dulbecco’s modified Eagle medium/Ham’s F-12 nutrient mix (DMEM/F12) (GibcoBRL 31300-038). The medium was supplemented with 6.7 µg/l sodium selenite, 10 mg/l insulin and 5.5 ml/1 transferrin (ITS- G, GibcoBRL 41400-045), 100 U/l penicillin/streptomycin (GibcoBRL 15140-114) and 1% serum Ultroser SF (Sopachem, France). Cells were cultured in 24-well plates (Greiner, The Netherlands) and seeded with 1 ml of cell suspension per well at 37 ℃ and 5% CO2. The culture medium was changed 24 h after plating, during which time the cells attached to the plate and reached almost confluency. Then the cells were exposed to test chemicals (brominated flame retardants), which were added to the wells at various concentrations using 2.5 ul of stock solutions dissolved in DMSO.

Enzymatic activity (CYP17) assay. The catalytic activity of CYP17 was determined after addition of 0.1 µM pregnenolone (precursor) to control and exposed H295R cells and measuring the production of DHEA using a

Table 1 Structures of brominated flame retardants (BFRs) and related compounds

Br (y)

PBDE Polybrominated diphenyl ether

2,4,6-TBP 2,4,6-tribromophenol

Br (x)

OH-BDE Hydroxy-bromodiphenyl ether

CH3

TBBPA Tetrabromobisphenol-A

CH3

OC3H5Br2

CH3

OC3H5Br2

TBBPA-DBPE Tetrabromobisphenol A - bis (2,3 dibromopropyl ether)

OCH3

CH3O-BDE Methoxy-bromodiphenyl ether

commercial RIA kit (Radioimmunoassay IM1138, Immunotech, Bechman Coulter Company). The inter-assay coefficient of variation was less than 10%. Pregnenolone metabolism into mineral and glucocorticoids via 3ß-hydroxyster- oid dehydrogenase mediation was inhibited with trilostane (1 µM). As a positive control for CYP17 inhibition, SU-10603 (1 µM) was used (Fig. 1).

Protein determination. The cells were stored at 4 ℃ for measurement of protein content within 2-4 days according to methods described earlier (Lowry et al., 1951; Rutten et al., 1987). Protein levels were determined from a standard curve that was generated using bovine serum albumin (Sigma A7030).

Cytotoxicity measurements. Cell viability, as an indicator of cytotoxicity, was determined by measuring the capacity of H295R cells (control and exposed) to reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan (Denizot and Lang, 1986). MTT is reduced to blue- colored formazan by the mitochondrial enzyme succinate dehydrogenase, which is considered a sensitive measure of mitochondrial function. The formazan formed in the cells was extracted by adding 1 ml of isopropanol and incubation for 10 min at room temperature. The alcohol fraction was measured spectrophotometrically at 595 nm (FLUOstar Galaxy V4.30-0/Stacker Control V1.02-0, BMG Labtechnologies).

Data analysis. All experiments were done in duplicated and within an indi- vidual experiment each concentration was tested in quadruplicate. Dose-response curves were drawn using Prism 3.0 (GraphPad Software Inc, USA). Results are presented as means with their standard deviations. Statistically significant differences from control groups were calculated using a two-tailed t test (P < 0.05).

Results

Enzymatic activity (CYP17) assay

The catalytic activity of CYP17 was determined based on the production of dehydroepiandrosterone (DHEA), a weak androgen produced by two CYP17 enzyme-mediated steps, 17a-hydroxylase and 17,20-lyase (see Fig. 1). After addition of 0.1 µM pregnenolone (precursor) in control and exposed H295R cells, the production of DHEA was measured using a radioimmunoassay (see Materials and methods). In order to measure CYP17 activity without interference of pregnen- olone metabolism into mineral and glucocorticoids, the enzyme 3ß-hydroxysteroid dehydrogenase was simultaneous- ly blocked with trilostane (1 µM). A pyridine derivative SU- 10603 (1 µM) was used as a positive control for human CYP17 inhibition (Fig. 1).

Preliminary experiments were carried out to establish the conditions of the CYP17 catalytic assay. Different concentra- tions of pregnenolone and the inhibitors trilostane and SU- 10603 concentrations as well as incubation times for the production of DHEA were studied for optimization (Figs. 2A and B). A range of pregnenolone concentrations was added to untreated H295R cells in order to measure DHEA production without interference from non-specific binding in the radioim- munoassay and 0.1 µM pregnenolone was chosen for subsequent experiments, when no cross-reactivity was mea- sured (<1%) (data not shown). The pyridine derivative SU- 10603 was chosen from a group of inhibitors (Fig. 2B) of the steroidogenesis pathway (imazalil, propiconazole, prochloraz and nonylphenol) because of its specificity for CYP17 (LaCagnin et al., 1989). No non-specific binding to SU-10603 (1 µM) was observed in the radioimmunoassay.

Fig. 1. Schematic diagram of the enzymatic activity (CYP17) assay. Trilostane and SU-10603, inhibitors of 3ß-hydroxysteroid dehydrogenase and CYP17 activity, respectively, were used at 1 µM.

Cholesterol

Trilostane (T) (inhibitor of 3ßHSD)

CYP11

Pregnenolone

3BH

Progesterone

17a-hydroxylase

(CYP17)

17a-hydroxylase (CYP17)

17a-hydroxypregnenolone

3BF

17a-hydroxyprogesterone

17,20 lyase

(CYP17)

17,20 lyase (CYP17)

Aromatase (CYP19)

estrone

Dehydroepiandrosterone (DHEA)

3115

androstenedione

Aromatase (CYP19)

SU-10603 (SU)

testosterone

17ß-estradiol

(inhibitor of CYP17 activity)

Effects of BFRs on CYP17 activity

BFRs were added to the cell cultures at concentrations ranging from 0.01 to 10 µM and possible inhibitory or inductive effects on CYP17 activity were determined after 24 h of exposure. Six BDEs were tested and, except BDE- 183, none of the congeners showed a significant effect on DHEA production in the H295R cells (Table 2). BDE-183 caused a moderate, but statistically significant inhibition of CYP17 of 20% of that of the controls at 10 µM. In addition, H295R cells were exposed to different phenolic brominated flame retardants (TBP, TBBPA, TBBPA-DBPE), but no inductive or inhibitive effect on CYP17 enzyme activity was measured either. At these concentrations no signs of cytotoxicity were observed with any of the BFR compounds tested (Table 2).

Effects of OH-BDEs derivatives on CYP17 activity

Initial range finding experiments with five different OH- BDEs (0.01 up to 10 µM) showed a significant inhibition, which was more than 50% of control CYP17 activity for 6- OH-BDE47, 6-OH-BDE99 and 4-OH-BDE49 at 10 µM (Table 2). 2-OH-BDE28 and 4-OH-BDE42 also showed a slight inhibition of CYP17 at the highest concentration tested, exceeding no more than 20-30% of the control (Table 2). However, cell viability measurements of MTT indicated that the decrease of CYP17 activity caused by 6-OH-BDE47, 4- OH-BDE49 and 2-OH-BDE28 occurred at cytotoxic concen- trations. In contrast, 6-OH-BDE99 and 4-OH-BDE42 were not found to be cytotoxic up to 10 µM, whereas for 6-OH-BDE99 a 67% inhibition of CYP17 activity could be found at 10 µM (Table 2).

Fig. 2. Optimization of CYP17 catalytic assay conditions. (A) Production of DHEA (as indicator of CYP17 activity) measured after 30, 45, 60 and 90 min of incubation time using pregnenolone (0.1 µM) as precursor, and the effects of trilostane (1 µM) (inhibitor of 3ß-HSD) and/or SU 10603 (1 µM) (inhibitor of CYP17 activity) in H295R human adrenocortical carcinoma cells. (B) CYP17 inhibition after treatment with SU-10603, propiconazole, prochloraz and nonylphenol at 1 µM. The graphs show one representative experiment from all experiments done in order to establish the best conditions for CYP17 assay.

CYP17 assay

30 min

# 45 min

DHEA (pg/mg prot/ml)

140000

60 min

120000

90 min

100000

80000

60000

40000

20000

Pregnenolone (0.1 μ.Μ)

Trilostane (1 µM)

SU-10603 (1 µM)

10 min

DHEA (ng/mg prot/ml)

D 20 min

0 90 min

Pregnenolone (0.1 µM) +++

+++

Trilostane (1 μM)

+++

SU-10603 (1 μM)

+++

Propiconazole (1 µM)

+++

Prochloraz (1 µM)

+++

Nonylphenol (1 µM)

+++

Table 2 Effects of selected brominated flame retardants (BFRs) and derivatives on CYP17 activity in human H295R adrenocortical carcinoma cells
Compound	CYP17 Inhibition % of control at 1 µM	CYP17 Inhibition % of control at 10 µM	MIT reduction % of control at uM
BDE-47 [2,2',4,4'-tetrabromodiphenyl ether]	116.55 ± 34.05	96.66± 10.06	99.05 ± 6.68
BDE-49 [2,2',4,5'-tetrabromodiphenyl ether]	106.39 ± 18.07	118.65 ±5.68	97.22 ± 12.71
BDE-99 [2,2',4,4',5-pentabromodiphenyl ether]	87.53 ± 35.28	102.48 ± 16.09	113.79 ±2.61
BDE-100 [2,2',4,4',6-pentabromodiphenyl ether]	100.71 ± 32.21	106.75 ± 47.70	102.06 ± 2.97
BDE-183 [2,2',3,4,4',5',6-heptabromodiphenyl ether]	116.82 ±20.73	80.3 ±12.28 (*)	106.55 ± 6.03
BDE-209 [2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether]	91.20 ± 30.37	89.34 ± 13.65	121.32 ± 56.2
2,4,6-TBP	116.02 ± 21.91	118.48 ±16.90	93.31 ± 2.25
TBBPA	117.40 ± 31.02	125.15 ± 32.54	96.73 ± 8.19
TBBPA-DBPE	117.44 ± 27.44	102.43 ± 10.11	94.29 ± 4.66
2'-OH-BDE28 [2',4,4'-tribromo-2-hydroxydiphenyl ether]	98.13 ±26.33	72.6 ± 15.20 (*)	66.26 ± 12.76 (*)
4'-OH-BDE42 [2,2',3,4'-tetrabromo-4-hydroxydiphenyl ether]	93.02 ± 29.76	79.5 ±20.90	79.77 ± 15.72
4'-OH-BDE49 [2,2',4',5-tetrabromo-4-hydroxydiphenyl ether]	88.15 ±24.58	41.5 ± 13.38 (*)	57.20 ± 7.27 (*)
4'-CH30-BDE49 [2,2',4',5-tetrabromo-4-methoxydiphenyl ether]	97.67 ± 25.34	90.44 ± 20.30	104.70 ± 5.7
6-OH-BDE99 [2',3,4,4',6-pentabromo-2-hydroxydiphenyl ether]	89.52 ±14.59	33.3 ± 11.94 (*)	97.26 ±26.58
6-CH3O-BDE47-BDE47 [2',4,4',6-tetrabromo-2-hydroxydiphenyl ether]	32.71 ± 16.92(*)	25.1 ± 12.05 (*)	10± 11.35 (*)
2'-OH-BDE47 [2',4,4',6'-tetrabromo-2-methoxydiphenyl ether]	83.06 ± 27.66	50.4 ± 20.99 (*)	105.34±22.01

Exposures were for 24 h in quadruplicate. Values represent means ± standard deviations (SD); n =2. * Significantly lower than control (P < 0.05).

Effects of CH3O-BDE derivatives on CYP17 activity

The methoxylated analogues of 6-OH-BDE47 and 4-OH- BDE49 were also tested to assess possible effects on CYP17 activity in the wider concentration range of 0.01-10 µM. 6- CH3O-BDE47 showed a statistically significant inhibition of the catalytic activity of CYP17 (of approximately 50%) at the highest concentration (10 µM) but not cytotoxicity (Fig. 3A). In contrast, 4-CH3O-BDE49 did not influence DHEA production

Fig. 3. Concentration-response curves for inhibition of CYP17 activity by 4- OH-BDE49, 6-OH-BDE47 and their methoxylated derivatives 6-CH3O- BDE47 and 4-CH3O-BDE49 treatments in the H295R human adrenocortical carcinoma cells. Exposures were for 24 h in quadruplicate. Values represent means ± standard deviations (SD) (n = 2). * Significantly lower than control (P < 0.05).

200

DHEA [pg/mg prot/h]

· 6CH30-BDE47

· 6OH-BDE47

₹

100

10-9

10-8

10-7

10-6

10-5

10-4

log [M]

200

· 4OH-BDE49

DHEA [pg/mg prot/h]

¥ 4CH30-BDE49

100

*HH

10-9

10-8

10- 7

10-

10-4 4

log [M]

at any of the concentrations used (Fig. 3B) nor were cytotoxic effects observed (Table 2).

Discussion

Detailed information about possible mechanisms of action is mostly lacking for these BFRs (e.g., TBBPA and PBDEs). However, both in vitro and in vivo experiments with, e.g., PBDEs and TBPPA, have shown that some of these compounds and/or their metabolites have effects on thyroid hormones, the estrogen receptor and neurobehavioral development (Meerts et al., 2001; Legler and Brouwer, 2003; Viberg et al., 2003a, 2003b). Based on these earlier results, certain BFRs are considered potential endocrine disruptors (Darnerud et al., 2001) for which more information is necessary with respect to mechanisms of action and concentration-effect relationships. Further, the penta-BDE mixture and some individual PBDEs did show anti-androgenic effects in several in vitro and in vivo studies (Stoker et al., 2004, 2005).

Previous studies in our laboratory found effects of several BFRs and their derivatives on aromatase (CYP19) activity in the H295R human adrenocortical carcinoma cell line. 2,4,6- Tribromophenol (TBP) showed a 4-fold induced on aromatase (CYP19) activity and several OH-PBDEs inhibited strongly aromatase (CYP19) activity. The presence and position of an OH group, or substitution with a CH3O group, had a modulating effect on the potency of the inhibition. Using the same cell line, it was also shown earlier that a number of herbicides, phytochemicals, fungicides and pesticides were capable of affecting aromatase (CYP19) activity (Sanderson et al., 2002; Heneweer et al., 2004).

In addition to aromatase, CYP17 enzyme also plays a key role in steroidogenesis by synthesizing weak androgens in the adrenal gland and the potent androgen testosterone in the testis. In the present study, fifteen different BFRs and their derivatives were studied in the H295R human adrenocarcinoma cell line to find potential interactions with CYP17. To measure this type of

steroidogenic interaction, a new bioanalytical method was developed with a radioimmunoassay for DHEA production measurements.

In our experiments, it was shown that none of the BFR parent compounds tested had an effect on CYP17 activity, except for some minor inhibition by BDE-183. However, CYP17 activity was inhibited significantly by almost all hydroxylated-PBDEs tested, most evidently by 4-OH-BDE49, 6-OH-BDE47 and 6- OH-BDE99. These hydroxylated-PBDEs caused as much as 75% reduction of the CYP17 activity at a concentration up to 10 uM for 6-OH-BDE47. However, this observed inhibitory effect by 6-OH-BDE47 and 4-OH-BDE49 can to some extent be explained by cytotoxicity due to the presence of an OH group in these molecules. This was illustrated by our experiments with the methoxy analogues 6-CH3O-BDE47 and 4-CH3O-BDE49 that did not show cytotoxicity. However, cytotoxicity by itself could not explain the effects of these compounds on CYP17 activity only, as in the case of 6-CH3O-BDE47 there was still significant inhibition but no cytotoxicity observed. In contrast, substitution of the OH group in 4-OH-BDE49 by a methoxy group caused a loss of CYP17 inhibitory properties. The reason for this congener-specific difference is presently unexplained, but the position of the OH is very likely to play a role. The significant role of the position of the (OH) hydroxyl as well as (CH3O) methoxy group possibly in combination with an adjacent bromine is also supported by the observations on cytotoxicity of 6-OH-BDEs, as 6-OH-BDE99 was found to be not cytotoxic whereas the other OH-BDE congeners were. Apparently hydroxylation and the 2 or 4 positions in the BDE molecule has a significant influence on cell viability. To our knowledge this is the first time that it is shown that metabolites of PBDEs can have CYP17 inhibiting capacity. Very few studies have addressed the possible interaction of environmental contaminants with CYP17. The inhibition of estradiol secretion was shown to be due to CYP17 inhibition when human luteinizing granulosa cells were treated with TCDD, which was not related to any decrease in aromatase activity (Moran et al., 2003).

Comparable results were reported earlier for the inhibitory effects of BFRs and some of their hydroxyl or methoxy analogues on CYP19 activity (Canton et al., 2005). Similar to our CYP17 study, the presence and position of an OH and CH3O group in a BDE congener influenced the potency of in vitro inhibition of CYP19 activity. The governing role of OH- versus CH3O groups in BDE substrates in inhibition of steroidogenic enzymes is apparently not restricted to BDEs alone. It has also been shown by Sanderson and coworkers (2004) who investigated the effects of various natural and synthetic flavonoids on the catalytic activity of aromatase in the H295R cell line. Some of these compounds (e.g., hydroxylated derivatives of flavonoids) were inhibitors of aromatase activity, whereas their methoxylated analogues mostly lacked aromatase inhibitory properties.

In addition, Lilienthal et al. (2006) showed effects on sex steroids in rats exposed to 2,2’,4,4’,5-pentabromoDE (BDE- 99). This study supports the hypothesis that PBDEs are also in vivo endocrine-active compounds and interfere with sex

steroidogenesis, sexual development and dimorphic behavior. Based on the results of our present and earlier in vitro studies (Canton et al., 2005), it can be discussed if the observed effects of BDE-99 (Lilienthal et al., 2006) were actually originating from hydroxyl-PBDE metabolites formed in vivo.

From an endocrinological point of view, the inhibition of CYP17 activity would result in a decrease of the synthesis of weak androgens, such as DHEA and consequently affect on testosterone and estradiol productions in testes and ovaries, respectively. DHEA is an abundantly produced adrenal steroid by CYP17 activity with pregnenolone being its precursor (Fig. 1). The biological role of DHEA so far has not been fully elucidated. Both in vitro and in vivo studies strongly indicate that DHEA can inhibit oxygen-free radical formation and at least partly inhibit related processes like inflammation, atherosclero- sis or carcinogenesis (Schwartz and Pashko, 2004). Already in the 1980s, in vivo studies have been done to elucidate the physiological role and properties of DHEA. In rodents, DHEA can cause numerous metabolic changes including a decrease in serum insulin as well as cholesterol levels (Cleary et al., 1984). In obese female mice, DHEA reduced weight gain without suppressing appetite. Furthermore, this steroid also antagonizes the formation of spontaneous mammary tumors in female mice (Schwartz, 1979). The results from the present study indicate a possible effect of OH- and CH3O-PBDEs on CYP17 activity and DHEA production in biota that could have endocrine con- sequences in mammals, including humans. Both OH- and CH3O-PBDEs have recently been reported to be present in various biotic samples including herring, salmon, seal and humans (Hovander et al., 2002; Marsh et al., 2004). In all these cases, OH- and CH3O-PBDE concentrations were measured within the pM and nM range; this is more than three order of magnitudes lower than the concentrations (1-10 µM range) that caused significant inhibition of CYP17 activity in our in vitro experiments. It should be noted that the occurrence of these OH- PBDEs in environmental samples can originate from either natural or anthropogenic sources (Marsh et al., 2004).

In conclusion, the biological significance and toxicological implications of the observed in vitro CYP17 inhibition by hydroxyl- or methoxylated-PBDEs needs further confirmation in vivo.

Acknowledgments

The work described in this paper was supported financially by the European Union under the “FIRE” project (contract number QLRT-2001-00596).

References

Alaee, M., Arias, P., Sjodin, A., Bergman, A., 2003. An overview of commercially used brominated flame retardants, their applications, their use patterns in different countries/regions and possible modes of release. Environ. Int. 29, 683-689.

Asplund, L., A.M., Sjodin, A., Bergman, A., Borjeson, H., 1999. Organohalo- gen substances in muscle, egg and blood from healthy Baltic salmon (Salmo salar) and Baltic salmon that produced offspring with the M74 syndrome. Ambio 28, 67-76.

Canton, R.F., Sanderson, J.T., Letcher, R.J., Bergman, A., van den Berg, M., 2005. Inhibition and induction of aromatase (CYP19) activity by brominated

flame retardants in H295R human adrenocortical carcinoma cells. Toxicol. Sci. 88, 447-455.

Chen, C.C., Parker Jr., C.R., 2004. Adrenal androgens and the immune system. Semin. Reprod. Med. 22, 369-377.

Ciolino, H., MacDonald, C., Memon, O., Dankwah, M., Yeh, G.C., 2003. Dehydroepiandrosterone inhibits the expression of carcinogen-activating enzymes in vivo. Int. J. Cancer 105, 321-325.

Cleary, M.P., Billheimer, J., Finan, A., Sartin, J.L., Schwartz, A.G., 1984. Metabolic consequences of dehydroepiandrosterone in lean and obese adult Zucker rats. Horm. Metab. Res. 16 (Suppl. 1), 43-46.

Darnerud, P.O., 2003. Toxic effects of brominated flame retardants in man and in wildlife. Environ. Int. 29, 841-853.

Darnerud, P.O., Eriksen, G.S., Johannesson, T., Larsen, P.B., Viluksela, M., 2001. Polybrominated diphenyl ethers: occurrence, dietary exposure, and toxicology. Environ. Health Perspect. 109 (Suppl. 1), 49-68.

de Wit, C.A., 2002. An overview of brominated flame retardants in the environment. Chemosphere 46, 583-624.

Denizot, F., Lang, R., 1986. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89, 271-277.

Gazdar, A.F., Oie, H.K., Shackleton, C.H., Chen, T.R., Triche, T.J., Myers, C.E., Chrousos, G.P., Brennan, M.F., Stein, C.A., La Rocca, R.V., 1990. Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res. 50, 5488-5496.

Gribble, G.W., 1996. Naturally occurring organohalogen compounds-a comprehensive survey. Fortschr. Chem. Org. Naturst. 68, 1-423.

Haglund, P.S., Zook, D.R., Buser, H .- R., Hu, J., 1997. Identification and quantification of polybrominated diphenyl ethers and methoxy-polybromi- nated diphenyl ethers in Baltic biota. Environ. Sci. Technol. 31, 3281-3287.

Hakk, H., Letcher, R.J., 2003. Metabolism in the toxicokinetics and fate of brominated flame retardants-a review. Environ. Int. 29, 801-828.

Hakk, H., Larsen, G., Klasson-Wehler, E., 2002. Tissue disposition, excretion and metabolism of 2,2’,4,4’,5-pentabromodiphenyl ether (BDE-99) in the male Sprague-Dawley rat. Xenobiotica 32, 369-382.

Heneweer, M., van den Berg, M., Sanderson, J.T., 2004. A comparison of human H295R and rat R2C cell lines as in vitro screening tools for effects on aromatase. Toxicol. Lett. 146, 183-194.

Hites, R.A., 2004. Polybrominated diphenyl ethers in the environment and in people: a meta-analysis of concentrations. Environ. Sci. Technol. 38, 945-956.

Hovander, L., Malmberg, T., Athanasiadou, M., Athanassiadis, I., Rahm, S., Bergman, A., Wehler, E.K., 2002. Identification of hydroxylated PCB metabolites and other phenolic halogenated pollutants in human blood plasma. Arch. Environ. Contam. Toxicol. 42, 105-117.

Kierkegaard, A., Bignert, A., Sellstrom, U., Olsson, M., Asplund, L., Jansson, B., De Wit, C.A., 2004. Polybrominated diphenyl ethers (PBDEs) and their methoxylated derivatives in pike from Swedish waters with emphasis on temporal trends, 1967-2000. Environ. Pollut. 130, 187-198.

Labrie, F., Luu-The, V., Labrie, C., Belanger, A., Simard, J., Lin, S.X., Pelletier, G., 2003. Endocrine and intracrine sources of androgens in women: inhibition of breast cancer and other roles of androgens and their precursor dehydroepiandrosterone. Endocr. Rev. 24, 152-182.

LaCagnin, L.B., Levitt, M., Bergstrom, J.M., Colby, H.D., 1989. Inhibition of adrenocortical, mitochondrial and microsomal monooxygenases by SU-10’ 603, a steroid 17 alpha-hydroxylase inhibitor. J. Steroid Biochem. 33, 599-604.

Law, R.J., Alaee, M., Allchin, C.R., Boon, J.P., Lebeuf, M., Lepom, P., Stern, G.A., 2003. Levels and trends of polybrominated diphenylethers and other brominated flame retardants in wildlife. Environ. Int. 29, 757-770.

Legler, J., Brouwer, A., 2003. Are brominated flame retardants endocrine disruptors? Environ. Int. 29, 879-885.

Lilienthal, H., Hack, A., Roth-Harer, A., Grande, S.W., Talsness, C.E., 2006. Effects of developmental exposure to 2,2,4,4,5-pentabromodiphenyl ether (PBDE-99) on sex steroids, sexual development, and sexually dimorphic behavior in rats. Environ. Health Perspect. 114, 194-201.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

Marsh, G., Hu, J., Jakobsson, E., Rahm, S., Bergman, Å., 1999. Synthesis and characterisation of 32 polybrominated diphenyl ethers. Environ. Sci. Technol 33, 3033-3037.

Marsh, G., Athanasiadou, M., Bergman, A., Asplund, L., 2004. Identification of hydroxylated and methoxylated polybrominated diphenyl ethers in Baltic Sea salmon (Salmo salar) blood. Environ. Sci. Technol. 38, 10-18.

Meerts, I.A., van Zanden, J.J., Luijks, E.A., van Leeuwen-Bol, I., Marsh, G., Jakobsson, E., Bergman, A., Brouwer, A., 2000. Potent competitive interactions of some brominated flame retardants and related compounds with human transthyretin in vitro. Toxicol. Sci. 56, 95-104.

Meerts, I.A., Letcher, R.J., Hoving, S., Marsh, G., Bergman, A., Lemmen, J.G., van der Burg, B., Brouwer, A., 2001. In vitro estrogenicity of polybrominated diphenyl ethers, hydroxylated PDBEs, and polybrominated bisphenol A compounds. Environ. Health Perspect. 109, 399-407.

Moran, F.M., Vande Voort, C.A., Overstreet, J.W., Lasley, B.L., Conley, A.J., 2003. Molecular target of endocrine disruption in human luteinizing granulosa cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin: inhibition of estradiol secretion due to decreased 17alpha-hydroxylase/17,20-lyase cytochrome P450 expression. Endocrinology 144, 467-473.

Morck, A., Hakk, H., Orn, U., Klasson Wehler, E., 2003. Decabromodiphenyl ether in the rat: absorption, distribution, metabolism, and excretion. Drug Metab. Dispos. 31, 900-907.

Noren, K., Meironyte, D., 2000. Certain organochlorine and organobromine contaminants in Swedish human milk in perspective of past 20-30 years. Chemosphere 40, 1111-1123.

Olsen, C.M., Meussen-Elholm, E.T., Holme, J.A., Hongslo, J.K., 2002. Brominated phenols: characterization of estrogen-like activity in the human breast cancer cell-line MCF-7. Toxicol. Lett. 129, 55-63.

Orn, U., Klasson-Wehler, E., 1998. Metabolism of 2,2’,4,4’-tetrabromodiphenyl ether in rat and mouse. Xenobiotica 28, 199-211.

Örn, U., Erikson, L., Jakobsson, E., Bergman, Å., 1996. Synthesis and characterisation of polybrominated diphenyl ethers-unlabeled and radiola- belled tetra-, penta- and hexabrominated diphenyl ethers. Acta Chem. Scand. 50, 802-807.

Ronisz, D., Finne, E.F., Karlsson, H., Forlin, L., 2004. Effects of the brominated flame retardants hexabromocyclododecane (HBCDD), and tetrabromobi- sphenol A (TBBPA), on hepatic enzymes and other biomarkers in juvenile rainbow trout and feral eelpout. Aquat. Toxicol. 69, 229-245.

Rutten, A.A., Falke, H.E., Catsburg, J.F., Topp, R., Blaauboer, B.J., van Holsteijn, I., Doorn, L., van Leeuwen, F.X., 1987. Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes. Reinvestigation of assay conditions. Arch. Toxicol. 61, 27-33.

Sanders, J.M., Chen, L.J., Lebetkin, E.H., Burka, L.T., 2006. Metabolism and disposition of 2,2’,4,4’-tetrabromodiphenyl ether following admin- istration of single or multiple doses to rats and mice. Xenobiotica 36, 103-117.

Sanderson, J.T., Seinen, W., Giesy, J.P., van den Berg, M., 2000. 2-Chloro-s- triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity? Toxicol. Sci. 54, 121-127.

Sanderson, J.T., Boerma, J., Lansbergen, G.W., van den Berg, M., 2002. Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol. Appl . Pharmacol. 182, 44-54.

Sandholm, A., Emanuelsson, B.M., Wehler, E.K., 2003. Bioavailability and half-life of decabromodiphenyl ether (BDE-209) in rat. Xenobiotica 33, 1149-1158.

Sanderson, J.T., Hordijk, J., Denison, M.S., Springsteel, M.F., Nantz, M.H., van den Berg, M., 2004. Induction and inhibition of aromatase (CYP19) activity by natural and synthetic flavonoid compounds in H295R human adrenocortical carcinoma cells. Toxicol. Sci. 82, 70-79.

Schwartz, A.G., 1979. Inhibition of spontaneous breast cancer formation in female C3H(Avy/a) mice by long-term treatment with dehydroepiandroster- one. Cancer Res. 39, 1129-1132.

Schwartz, A.G., Pashko, L.L., 2004. Dehydroepiandrosterone, glucose-6- phosphate dehydrogenase, and longevity. Ageing Res. Rev. 3, 171-187.

Sellstrom, U., Bignert, A., Kierkegaard, A., Haggberg, L., de Wit, C.A., Olsson, M., Jansson, B., 2003. Temporal trend studies on tetra- and pentabrominated

diphenyl ethers and hexabromocyclododecane in guillemot egg from the Baltic Sea. Environ. Sci. Technol. 37, 5496-5501.

Stapleton, H.M., Alaee, M., Letcher, R.J., Baker, J.E., 2004. Debromination of the flame retardant decabromodiphenyl ether by juvenile carp (Cyprinus carpio) following dietary exposure. Environ. Sci. Technol. 38, 112-119.

Staskal, D.F., Diliberto, J.J., De Vito, M.J., Birnbaum, L.S., 2005. Toxicokinetics of BDE 47 in female mice: effect of dose, route of exposure, and time. Toxicol. Sci. 83, 215-223.

Stoker, T.E., Laws, S.C., Crofton, K.M., Hedge, J.M., Ferrell, J.M., Cooper, R. L., 2004. Assessment of DE-71, a commercial polybrominated diphenyl ether (PBDE) mixture, in the EDSP male and female pubertal protocols. Toxicol. Sci. 78, 144-155.

Stoker, T.E., Cooper, R.L., Lambright, C.S., Wilson, V.S., Furr, J., Gray, L.E., 2005. In vivo and in vitro anti-androgenic effects of DE-71, a commercial polybrominated diphenyl ether (PBDE) mixture. Toxicol. Appl. Pharmacol. 207, 78-88.

Thomas, G.O., Moss, S.E., Asplund, L., Hall, A.J., 2005. Absorption of decabromodiphenyl ether and other organohalogen chemicals by grey seals (Halichoerus grypus). Environ. Pollut. 133, 581-586.

Thuresson, K., Bergman, A., Jakobsson, K., 2005. Occupational exposure to commercial decabromodiphenyl ether in workers manufacturing or handling flame-retarded rubber. Environ. Sci. Technol. 39, 1980-1986.

Valters, K., Li, H., Alaee, M., D’Sa, I., Marsh, G., Bergman, A., Letcher, R.J., 2005. Polybrominated diphenyl ethers and hydroxylated and methoxylated brominated and chlorinated analogues in the plasma of fish from the Detroit River. Environ. Sci. Technol. 39, 5612-5619.

Viberg, H., Fredriksson, A., Eriksson, P., 2003a. Neonatal exposure to polybrominated diphenyl ether (PBDE 153) disrupts spontaneous behaviour, impairs learning and memory, and decreases hippocampal cholinergic receptors in adult mice. Toxicol. Appl. Pharmacol. 192, 95-106.

Viberg, H., Fredriksson, A., Jakobsson, E., Orn, U., Eriksson, P., 2003b. Neurobehavioral derangements in adult mice receiving decabrominated diphenyl ether (PBDE 209) during a defined period of neonatal brain development. Toxicol. Sci. 76, 112-120.

Zhou, T., Taylor, M.M., DeVito, M.J., Crofton, K.M., 2002. Developmental exposure to brominated diphenyl ethers results in thyroid hormone disruption. Toxicol. Sci. 66, 105-116.

Quartz 4

Explorer

16828825