Chitose Toda

Effects of phthalate ester derivatives including oxidized metabolites on coactivator recruiting by PPARα and PPARγ

Phthalate esters (PEs), a group of environmental chemicals, affect biological systems via endocrine and lipid metabolism modulations. These effects are believed to be mediated in part by peroxisome proliferator-activated receptors (PPARs). Evaluations of PE activities as ligands toward PPARs have been investigated in many studies on their primary metabolites, monoesters. However, the activities of various other metabolites, including oxidized derivatives, remain to be determined. Here, we have evaluated the PPAR ligand activities of these PE derivatives by in vitro coactivator recruiting assay. Mono(2-ethyl-5-hydroxyhexyl)phthalate, the most abundant metabolite of di-(2-ethylhexyl)phthalate (DEHP), was less active than mono(2-ethylhexyl)phthalate (MEHP) as a PPAR ligand. Other derivatives oxidized at the alkyl group and benzene ring of DEHP, MEHP, dibutyl phthalate and its monoester were also investigated and some affected PPAR activities. Unexpectedly, MEHP as well as its further oxidized metabolite did not show clear activity for PPARalpha, although MEHP is believed to interact with PPARalpha. This might imply indirect PPAR-mediated mechanisms that lead to observed biological effects such as peroxisome proliferation.

Structural properties of estrogen receptor ligand obtained by study of hydroxylated phthalate ester derivatives.

Phthalate esters (PEs), a group of plasticizers, are suspected to be endocrine-disrupting chemicals. Here, PE derivatives were used as probes for elucidating the structural properties of estrogen receptor (ER) ligands. A comprehensive study was performed using more than 40 PE derivatives including ring-/alkyl-hydroxylated and nonsymmetrical diesters possessing independently altered alkyls of C1-C8. Estrogenic activity of these derivatives is determined with three assays for ER-binding, coactivator-recruiting and transactivation. Phenolic hydroxylation increased activity, while hydroxylation of the ester alkyl group had no distinct effect on ER binding or transcription coactivator recruitment. Ring-hydroxylated PE derivatives harboring different ester alkyls revealed that the length of both alkyls independently affects transactivation of ER. These comprehensive data would be useful for the better understanding of structure-activity relationship of endocrine-disrupting chemicals.

Determination of Butylated Hydroxyanisole and Its Conjugated Metabolites in the Organs, Blood and Excreta of Mice by High-Performance Liquid Chromatography

The determination of butylated hydroxyanisole (BHA) and its conjugated metabolites (glucuronide and sulfate) in tissues and excreta of BHA administered mice was investigated by normal-phase high-performance liquid chromatography (HPLC). Male mice were orally administered BHA at 50 and 500 mg/kg, BHA and its metabolites were extracted from the liver and kidney by continuous extraction and from the blood, stomach, intestine, urine and feces by direct extraction. Conjugated metabolites were hydrolyzed with enzymes before extraction. BHA could be determined without influence from obstructive substances. In animals administered BHA at 500 mg/kg, unchanged BHA was present at high concentration in the liver, kidney and the blood 0.5 h later, but at 8 h could not be detected in any sample. The sulfate of BHA exceeded the glucuronide of BHA in the liver at 0.5 h, but the latter exceeded the former during 1 and 3 h after dosing. Mice excreted about 52% of 500 mg/kg during 8 h, and about 76% within 48 h in the urine as glucuronide (72.3±7.6%), sulfate (3.0±2.4%) and unchanged BHA (0.3±0.1%). However, about 25% of unchanged BHA was recovered from stomach at 8 h, and even after 48 h. some unchanged BHA could be found.