Mechanism of Action of Brominated Flame Retardants

Transcription

1 S R A nstitute for Risk Assessment Sciences Æ Mechanism of Action of ominated Flame Retardants mplications for risk assessment Martin van den Berg nstitute for Risk Assessment Sciences, Utrecht University, World Health Organization Collaborating Centre for Research on Environmental Health Risk Assessment PO Box 80176, 3508 D, Utrecht, he Netherlands EU-FRE project QK4-C omine Scientific and Environmental Forum (BSEF)

2 S R A Research Scientists Æ homas Sanderson Ass. Professor ieke Peters Ah Receptor interactions mammalian systems Raoul Kuiper Ah Receptor interactions fish systems Rocio Fernandez Endocrine disruption via steroidogenesis

3 S R A Æ Polybrominated diphenylethers - Dioxin like compounds and interaction with PCBs and dioxins? - Several PBDEs are persistent in the abiotic environment Some of these PBDEs bioaccumulate (e.g. PBDEs 47, 99, 100, 153 and 154) evels of these PBDEs increased in fish, wildlife and human tissues/milk during last decades

4 S R A Æ Most common PBDEs in environment and biota Ortho substituted pattern! O O O O r DE47 BDE77 (Dioxin like?) BDE153 BDE154 O O O O BDE99 BDE100 BDE183 BDE209

5 S R A Æ Research Questions? Do certain PBDEs have dioxin like activity mediated via the Ah-receptor? f so, should PBDEs be included in the EF concept?

6 S R A Æ Contamination PBDEs? Cause for dioxin like effects? Prof Ake Bergman Stockholm University Prof Robert etcher University Windsor

7 S R A Æ CDD AhR AhR Heat shock proteins oxicity EROD activity PBDEs?? Cytoplasm CYP1A1, 1A2,1B1 Arnt Prot Nucleus mrnas DRE Dioxin responsive Gene

8 S R A Æ PBDE s dioxin like compounds in mammalian and fish systems?

9 Methods Cell systems (dioxin responsive) Human breast carcinoma MCF-7 cells Human hepatoma HepG2 cells Rat hepatoma H4E cells Carp hepatocytes (liver cells) Measured parameters: CYP1A1 mediated EROD activity CYP1A1 mrna Positive control CDD S R A Æ

10 Results EROD (CYP1A1) assay mammalian cell systems most common PBDEs resorufin/min/mg protein) 350 EROD activity in HepG2 cells Human liver cancer cells Concentration (log M) CDD PBDEs EROD activity (pmol resorufin /min /mg protein) 80 EROD activity MCF7 cells Human breast cancer cells Concentration (log M) CDD PBDEs 47,99,100, 153,154,183, EROD activity in H4e cells Rat liver cancer cells CDD PBDEs ow solubility! Concentration (log M)

11 he Carp hepatocyte assay A method for measuring dioxin like and (anti) estrogenicity activity oxicol. Appl. Pharmacol. 157,68-76 (1999) ox. Sciences 50, (1999) ox. Sciences 53, (1999) S R A

12 S R A Carphep Endocrine Functions Dioxins CYP1A (EROD) Æ

13 S R A Æ

14 S R A ominated flame retardants tested in the Carp hepatocytes Nr Compound Mw EROD G cytotox 1 BDE 19 [2,2,6] BDE 28 [2,4,4 ] ?? 3 BDE 38 [3,4,5] ?? 4 BDE 39 [3,4,5] BDE 47 [2,2,4,4 ] BDE 49 [2,2,4,5 ] BDE 79 [3,3,4,5 ] BDE 99 [2,2,4,4,5] BDE 100 [2,2,4,4,6] BDE 127 [3,3,4,5,5 ] BDE 153 [2,2,4,4,5,5 ] BDE 155 [2,2,4,4,6,6 ] BDE 169 [3,3,4,4,5,5 ] BDE 181 [2,2,3,4,4,5,6] BDE 183 [2,2,3,4,4,5,6] BDE 185 [2,2,3,4,5,5,6] BDE 190 [2,3,3,4,4,5,6] BDE 206 [2,2,3,3,4,4,5,5,6] BDE 209 [2,2,3,3,4,4,5,5,6,6 ] ? 20 BBPA (Aldrich) ? 21 BBPA (RM) ? 22 2,4,6-tribromophenol OH-BDE HBCDD technical mixture HBCDD alpha isomer HBCDD beta isomer HBCDD gamma isomer BBPA-DBPE (FR-720) allylether-bbpa 30 2,3-dibromopropylether-BBPA 31 tetrabromo-diethyl phtalate ester + ~loec; ++>20% max; all p-values<0.01

15 S R A Æ CYP1A induction resorufin prod. (pmol/well/min) CDD 5-6 orders of magnitude difference with CDD CDD BDE-38 BDE-79 BDE-183 BDE og concentration (M) Some PBDEs appear to induce CYP1A in Carp hepatocytes Difference with CDD 5 to 6 orders of magnitude in potency he most environmental relevant PBDEs do not significantly induce CYP1A

16 S R A Æ Conclusions PBDEs Ah-Receptor mediated interaction? he most environmental common PBDEs do not appear to act significantly through the AhR when extensively purified in both mammalian and fish cell systems that are dioxin responsive he absence or very low AhR mediated response indicates that PBDEs do not have to be included in the EF concept for dioxin like compounds Previous dioxin like results with PBDEs might have been caused by presence of polybrominated dibenzofuran contamination.

17 S R A Æ Research Question? Could PBDE congeners be (weak) AhR antagonists due to resemblance to di-ortho-pcbs e.g. PCB153?

18 S R A Æ Results MCF-7 Cells EROD activity (pmol resorufin /min /mg protein) EROD activity MCF7 cells Human breast cancer CDD CDD+BDE153 1µMl CDD+BDE153 10µM 60 CDD+BDE77 1µM CDD+BDE77 10µM Concentration (log M) Approximately 30 to 50% reduction of CYP1A1 activity by PBDEs 77 and 153 at 10 um at maximum induced EROD activity by CDD

19 S R A Æ Similar results HepG2 and H4E cells EROD activity in HepG2 cells Human liver CDD CDD+BDE153 1µM CDD+BDE153 10µM Co ncentratio n (lo g M) Approximately 25% reduction of CYP1A1 activity by PBDEs 153 at 10 um at maximum induced EROD activity by CDD. EROD activity (pmol sorufin/min/mg protein) re EROD activity in H4e cells Rat liver Concentration (log M) CDD CDD+BDE153 1µM CDD+BDE153 10µM Dioxin Conference 2003 Other PBDEs show similar reduction in EROD activity in these cell lines but quantitative differences in reduction are observed.

20 25 BDE BDE-100 EROD (pmol/mg/min EROD (pmol/mg/min concentration CDD (pm) 0 µm BDE µm BDE µm BDE-47 Carphep assay concentration CDD (pm) 0 µm BDE µm BDE µm BDE-100 PBDEs caused a strong congener dependent decrease in the Dioxin like induced CYP1A induction via the Ah receptor in the Carphep assay at similar ratio s as in the mammalian systems 25 BDE-99 BDE-153 EROD (pmol/mg/min concentration CDD (pm) 0 µm BDE µm BDE µm BDE-99 EROD (pmol/mg/min concentration CDD (pm) 0 µm BDE µm BDE µm BDE-153

21 S R A Æ Conclusions At maximum CDD-induced CYP1A (EROD) activity these PBDEs reduce this type of AhRmediated activity by as much as 50% or more in both mammalian and fish system Ratio s 1 to for dioxin to PBDEs at maximum induced level (realistic for environmental biota?) Does it have implications for dioxin toxicity?

22 Effects of brominated flame retardants (BFRs) on activity of the steroidogenic enzyme aromatase (CYP19) in H295R human adrenocortical carcinoma cells in culture S R A Æ

23 S R A Æ ominated diphenylethers: Several in vitro studies showed how these compounds, especially some metabolites, have endocrinological effects (e.g. thyroidhormone and weak estrogen receptor interaction, mostly in vitro) Endocrine disrupting chemicals? At receptor level (estrogen, androgen, glucocorticoid etc.) At enzyme level (effects in the catalytic activity of the enzyme or in the enzyme expression) in steroidogenesis

24 S R A Steroidogenesis: Æ adrenals Cholesterol CYP11A Pregnenolone 3βHSD Progesterone 17α-hydroxylase (CYP17) 17α-hydroxylase (CYP17) 17α-hydroxypregnenolone 3βHSD 17α-hydroxyprogesterone 17,20 lyase (CYP17) 17,20 lyase (CYP17) Dehydroepiandrosterone (DHEA) 3βHSD androstenedione testosterone Aromatase (CYP19) Aromatase (CYP19) estrone 17β-estradiol e.g. ovaries and breast tissue 5α-reductase Dihydrotestosterone (DH) e.g. testis

25 S R A Æ Conversion of androgens to estrogens by Aromatase activity (CYP19) CH 3 O C H 3 O H H CH 3 CYP19 O HO Androstenedione estosterone Estrone Estradiol Expression important for development, reproduction, behaviour and hormone dependent tumors

26 S Æ Nr Compound 1 BDE 19 [2,2,6] 2 BDE 28 [2,4,4 ] 3 BDE 38 [3,4,5] 4 BDE 39 [3,4,5] 5 BDE 47 [2,2,4,4 ] 6 BDE 49 [2,2,4,5 ] 7 BDE 79 [3,3,4,5 ] 8 BDE 99 [2,2,4,4,5] 9 BDE 100 [2,2,4,4,6] 10 BDE 127 [3,3,4,5,5 ] 11 BDE 153 [2,2,4,4,5,5 ] 12 BDE 155 [2,2,4,4,6,6 ] 13 BDE 169 [3,3,4,4,5,5 ] 14 BDE 181 [2,2,3,4,4,5,6] 15 BDE 183 [2,2,3,4,4,5,6] 16 BDE 185 [2,2,3,4,5,5,6] 17 BDE 190 [2,3,3,4,4,5,6] 18 BDE 206 [2,2,3,3,4,4,5,5,6] 19 BDE 209 [2,2,3,3,4,4,5,5,6,6 ] 20 BBPA (Aldrich) 21 BBPA (RM) 22 FR ,4,6-tribromophenol 24 HBCDD technical mixture 25 HBCDD alpha isomer 26 HBCDD beta isomer 27 HBCDD gamma isomer OH-BDE 47 2OHBDE ested brominated flame retardants for aromatase interaction 4OHBDE42 6OHBDE99 4OHBDE49 4MeOBDE49 O HO (OH) CH 3 CH 3 OH O OH PBDE metabolites ntermediate and Natural product

27 S R A Æ Effects of BFRs on aromatase in H295R cells aromatase activity(% of control) control BDE28 BDE47 BDE49 BDE99 BDEs BDE100 BDE153 BDE155 BDE microm 7.5 microm BDE183 BDE190 BDE 28 BDE 47 BDE 49 BDE 99 BDE 100 BDE 153 BDE 155 BDE 181 BDE 185 BDE 190 Similar response for these BFRs, no effect No cytotoxicity 2.5 microm aromatase activity (%of control) 140,00 120,00 100,00 80,00 60,00 40,00 20,00 0,00 * control BBPA FR-720 HBCDD 7.5 microm BBPA FR-720 HBCDD nhibition for FR-720 (tetrabromobisphenol substitute)

28 nhibition of aromatase activity by some hydroxylated PBDEs metabolites aromatase activity(% of control) control 2OH BDE28 4OH BDE42 * 6OH BDE47 4OH BDE49 * 6OH BDE microm 7.5 microm 2 OH BDE 28 4 OH BDE 42 6 OH BDE 47 4 OH BDE 49 6 OH BDE 99 Data suggest some OHBDEs (47 and 99 with 6 OH group) can decrease aromatase activity significantly f relevant for in vivo situation this might lead to antiestrogenic or androgenic effects S R A Æ

29 S R A N O Æ Aromatase inhibition by 6OH-BDE47 due to cytotoxicity? aromatase activity (% of control) * * * * control 0,75 1,25 2 2,5 3,75 5 7,5 microm 6OHBDE47 After 24h.exposure to 6OHBDE47 aromatase activity decreased 95% at 7.5 µm C 50 = 3.6 µm Cytotoxicity assays indicate that this PBDE metabolite has an adverse effect on mitochondrial respiration and plasma membrane integrity. his could (in part) explain the inhibition of aromatase in these human cells

30 S R A N O Æ O aromatase activity (% of control) aromatase activity (% of control) OHBDE47 * * * control microm MeOBDE47 * * control microm s the 6-OH group playing a dominant role in cytotoxicity and aromatase inhibition? Switch 6 OH with 6 OCH 3 group and no cytotoxicity found, but 50 % aromatase inhibition still present Metabolites of PBDEs might have some inhibitive effect on aromatase besides general cytotoxicity

31 S R A Æ Do bromine atoms in combination with OH group have influence on aromatase? 6OH-BDE47 O HO nhibition of aromatase activity High cytotoxicity with damage plasma membrane 6OH-BDE99 O HO nhibition of aromatase activity No cytotoxicity effects Adjacent bromine appears to reduce cytotoxicity of 6 OH group in BDEs

32 nduction of aromatase activity by 2,4,6-tribromophenol in H295R cells OH aromatase activity (% of control) control 0,5 1,25 2,5 3,75 5 7,5 microm tribromophenol he activity of aromatase in the H295R cells was concentration dependently increased 4-fold above control S R A Æ

33 S R A N O Æ How structure specific is induction of aromatase by tribromophenol? OH OMe ribromophenol OH ribromoanisole (Substitution by methoxygroup) 4-bromophenol (removal bromines)

34 No effects of tribromoanisole and 4-bromophenol on aromatase in H295R cells 140 OH aromatase activity (% of control) OMe 0 control 2.5 microm 5 microm 7.5 microm Combination with OH group and adjacent bromine atoms seems required for induction of aromatase S R A Æ

35 S R A Æ Conclusions he parent compounds of PBDEs have no influence on in vitro aromatase activity Hydroxylated BFRs show more biological or toxicological activity in human H295R cells 6 OH metabolites of PBDEs can be highly cytotoxic Both OH and groups play a role in in vitro induction of aromatase activity