Human hydroxylated metabolites of BDE-47 and BDE-99 are glucuronidated and sulfated in vitro

• This is the first study investigating human Phase-II metabolism of PBDEs in vitro.
• All hydroxylated metabolites of BDE-47 and BDE-99 were glucuronidated and sulfated.
• 2,4-DBP-Gluc and 5-Gluc-BDE-47 are the major glucuronidated metabolites of BDE-47.
• 2,4,5-TBP-Gluc and 6′-Gluc-BDE-99 are the major glucuronidated metabolites of BDE-99.
• 4-Sulf-BDE-42 and 3-Sulf-BDE-47 are the major sulfated metabolites of BDE-47.
• 3′-Sulf-BDE-99 and 5′-Sulf-BDE-99 are the major sulfated metabolites of BDE-99.

Polybrominated diphenyl ethers (PBDEs) were used worldwide as additive flame retardants and are classified as persistent, bioaccumulable and toxic environmental pollutants. In humans, the hydroxylated metabolites of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99) formed in vitro have also been detected in vivo. To further characterize the metabolism of BDE-47 and BDE-99 and to identify candidate markers for monitoring the human exposure to PBDEs using non-invasive approaches, glucuronidation and sulfation of hydroxylated metabolites of BDE-47 and BDE-99 were investigated using human liver microsomes and cytoplasm, respectively. The formed Phase II metabolites were analyzed by liquid chromatography-tandem mass spectrometry using a novel approach to develop analytical methods in absence of authentic standards. All available standards for hydroxylated metabolites of BDE-47 and BDE-99 were glucuronidated and sulfated, showing that glucuronidation and sulfation are part of the metabolism pathway of BDE-47 and BDE-99 in vitro. The major glucuronidated and sulfated analogs of hydroxylated metabolites of BDE-47 were (a) 2,4-DBP-Gluc and 5-Gluc-BDE-47, and (b) 2′-Sulf-BDE-28, 4-Sulf-BDE-42 and 3-Sulf-BDE-47, respectively. The major glucuronidated and sulfated analogs of hydroxylated metabolites of BDE-99 were (a) 2,4,5-TBP-Gluc and 6′-Gluc-BDE-99, and (b) 3′-Sulf-BDE-99 and 5′-Sulf-BDE-99, respectively. Apparent Km values associated with the formation of sulfated metabolites of BDE-47 and BDE-99 were ten times lower than those of the corresponding glucuronidated metabolites, suggesting that sulfated rather than glucuronidated metabolites of OH-PBDEs might be used as markers of human exposure to PBDEs using a non-invasive approach based on urine sample collection.