Teratogenic phthalate esters and metabolites activate the nuclear receptors PPARs and induce differentiation of F9 cells

Industrial plasticizers such as phthalates can induce peroxisome proliferation. Some phthalates such as di-2-ethyl-hexyl-phthalate (DEHP) and its metabolites mono-2-ethyl-hexyl-phthalate and 2-ethyl-hexanoic acid are also known teratogens. Recently, we introduced two in vitro test systems consisting of F9 teratocarcinoma cell differentiation and activation of peroxisome proliferator-activated receptor (PPAR)-ligand-binding domain in Chinese hamster ovary-reporter cells for the detection of teratogenic compounds related to the antiepileptic drug valproic acid. We now applied these methods to the class of phthalate esters and their metabolites by testing 2 diphthalate esters and 19 monophthalate esters in vitro. In the F9 cell assay only five compounds, mono-2-ethyl-hexyl-phthalate, mono-1-methyl-heptyl-phthalate, mono-benzyl-phthalate, benzyl-butyl-phthalate, and 2-ethyl-hexanoic acid were found to induce F9 cell differentiation. The other test compounds were not able to induce differentiation of F9 cells. Three compounds (mono-methyl-phthalate, mono-ethyl-phthalate, and mono-2,2-dimethyl-1-phenyl-propyl-phthalate, and phthalic acid di-methyl-ester were found not to interact with any PPARs. All other phthalate esters activated PPARs. Most compounds activated PPARalpha and PPARgamma. Interestingly PPARgamma in most cases was activated stronger than PPARalpha. Only the five test compounds, mono-2-ethyl-hexyl-phthatate, mono-1-methyl-heptyl-phthalate, mono-benzyl-phthalate, benzyl-butyl-phthalate, and 2-ethyl-hexanoic acid activated PPARdelta and interacted with a specific PPARdelta-response element. These are the same compounds that induced F9 cell differentiation and three of them are known teratogenic compounds. It is concluded that phthatate esters are acting like hormones by activating PPARs. The combination of F9 cell differentiation assay and PPARdelta activation assay detected possible teratogenic phthalate-ester and derivatives. Therefore the test systems seem useful for a screening test system in the early development of new plasticizers.