Brominated Flame Retardants and their metabolites: Novel insights into endocrine disruptive properties.

Brominated flame retardants (BFRs) are chemicals that are added to materials to inhibit or suppress ignition and are incorporated during the manufacture of e.g. electronic equipment, furniture, construction materials and textiles. BFRs have become an increasingly important group of organohalogen compounds, which include hexabromocyclododecane diastereomers (HBCDs), tetrabromobisphenol-A (TBBPA) and commercial mixtures of polybrominated diphenyl ether (PentaBDE, OctaBDE and DecaBDE) congeners. The use of BFRs has increased tremendously during the last decades mainly due to the high demand of petroleum-based polymeric materials which have to meet strict fire safety regulations to prevent possible accidents. There are five major classes of BFRs: tetrabromobisphenol-A (TBBPA), hexabromocyclododecane (HBCD), and three commercial mixtures of polybrominated diphenyl ethers (PBDEs), or biphenyl oxides, which are known as pentabromodiphenyl ether (PentaBDE), octabromodiphenyl ether (OctaBDE) and decabromodiphenyl ether (DecaBDE). However, OctaBDE and PentaBDE are not longer being produced or in use due to their ban in EU and voluntary withdrawal by the bromine industry in North America in 2004. The main purpose of this thesis is to assess the in vitro effects of brominated flame retardants and their metabolites on sex steroid hormone action. Sex steroid hormones, such as, estrogens or androgens are formed by steroidogenic enzymes in several endocrine glands. Two of the most relevant steroidogenic enzymes are the cytochrome P450, CYP17 and CYP19 (also known as aromatase). Using the human adrenocortical carcinoma H295R cell line, the potential of BFRs (PBDEs, TBBPA and HBCD) and some of their metabolites (hydroxylated and methoxylated PBDEs) to alter aromatase activity was analyzed. Development and validation of an in vitro enzymatic technique to study the possible effects on CYP17 activity by these compounds is included in the present thesis. In general, parent PBDEs did not significantly alter CYP19 or CYP17 activities, but pronounced inhibitory effects were found for their hydroxylated metabolites, although to some extend these effects were due to cytotoxicity. In order to differentiate between cytotoxic effects of these hydroxylated metabolites and actual catalytic inhibition of aromatase, a series of experiments with human placental microsomes were performed. All hydroxylated metabolites tested showed a significant inhibition of aromatase activity. Furthermore, the agonistic and antagonistic properties on the human androgen receptor (AR) by several BFRs and PBDE metabolites were studied in two different in vitro recombinant systems. No agonistic effects on the AR were detected by any of the tested compounds, although several of the BFRs and hydroxylated PBDEs exhibited anti-androgenic activity in both recombinant in vitro systems. Furthermore, two genomic techniques (microarray and qRT-PCR) were used to describe the changes in hepatic gene profiles in HBCD exposed rats. The results induced clear sex specific changes in the hepatic gene expression profiles, suggesting female rats to be more sensitive. The effects presented in this thesis are in good agreement with previous in vitro, as well as, in vivo studies and novel information about the mechanism of action of these BFRs and PBDE metabolites were elucidated.