Effects of phthalate ester derivatives including oxidized metabolites on coactivator recruiting by PPARalpha and PPARgamma.

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.

Formation of estrogenic products from environmental phthalate esters under light exposure.

Phthalate esters (PEs) have been suspected to be environmental endocrine disruptors and the detailed mechanism remains unclear. The activities of these chemicals can be enhanced through chemical modification under the environmental conditions. We demonstrate that PEs acquire unequivocal estrogenic activity by light exposure. Through UV exposure of an aqueous PE solution, one active photoproduct, identified as 4-hydroxyPE (PE-4OH) based on its characteristic UV and mass spectra, was detected in an estrogen receptor alpha-dependent transactivation assay. PE-4OH was effectively generated by UV 290 nm. The PE-4OH production accompanied H2O2 generation in a UV dose-dependent manner. Both PE and UV irradiation were indispensable in the generation of H2O2. Addition of H2O2 to the PE solution increased PE-4OH production under UV irradiation. The PE-4OH production was also observed in the PE reaction with the Fenton reagent generating hydroxyl radical without UV irradiation. The proposed mechanism for PE-4OH production based on these results is such that by PE-mediated photosensitization H2O2 is generated from O2 and H+ and decomposed to hydroxyl radical, thus oxidizing the PE benzene ring. The PEs-4OH are remarkably active estrogenic products of PEs and would be involved in ER-mediated endocrine disruption.

Unequivocal estrogen receptor-binding affinity of phthalate esters featured with ring hydroxylation and proper alkyl chain size.

The effect of plasticizers phthalate esters (PEs) on health is a controversial subject. PEs are likely to be estrogenic, but the results on the potency obtained by many investigators are still inconsistent and the endocrine disrupting mechanism remains to be clarified. Here, we show that PEs acquire unequivocal binding affinities for human estrogen receptors (ERs) through ring hydroxylation that is possible in the environment and through metabolism. Unexpectedly, the acquired affinities of hydroxylated PEs (PEs-OH) were enhanced by elongation and branching of the ester alkyl chains. PEs-OH with alkyl chains more than six carbons may grope for a new binding site, which is inaccessible to PEs-OH with short chains. The strongest ER-binding affinity among the tested PEs-OH was close to that of diethylstilbestrol, the most potent synthetic ER-binder. Ring hydroxylation would be a new clue to the clarification of the endocrine disruption mechanism of PEs.

Phthalate esters detected in various water samples and biodegradation of the phthalates by microbes isolated from river water.

Phthalate esters (PEs), especially di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) were detected in various water samples such as river water, well water and tap water. On degradation tests of PEs, Tempaku River water degraded almost 100% of diethyl phthalate (DEP), di-isobutyl phthalate and DBP, and approximately 70% of DEHP. All eight isolates from Tempaku River water (R1-R7, D1) did not degrade dimethyl phthalate (DMP), but showed biodegrading ability for the other PEs. The DBP-degrading ability was particularly high for the isolates R1-R3 and D1 of Acinetobacter iwoffii. Crude enzyme solutions prepared from bacterial cells of these isolates showed a higher degrading activity for DEHP compared with that for microbially-degradable DBP. Particularly high DEHP-degrading activity was found for crude enzyme solutions of the isolate D1. As metabolites from the river water and bacterial isolates, DMP and an unknown diester were produced from DEP. DMP, DEP, monomethyl phthalate, monobutyl phthalate (MBP) and an unknown diester were produced from DBP. DBP, DEP, DMP and an unknown diester were produced from DEHP. As metabolites by the crude enzyme solutions, DMP, MBP and an unknown diester derivative were produced from DBP. DBP, mono-(2-ethylhexyl) phthalate and an unknown diester derivative were produced from DEHP. Diesters with shortened alkyl carbon chains were also found as metabolites by the isolates and their crude enzyme solutions. The results suggest that the alkyl chains in the diesters are also decomposed in addition to monoester formation from DBP or DEHP at the first step reported for animals and some types of bacteria.