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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), tetrabromobisphenol-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 17α-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.