Evaluation of a predictive in vitro Leydig cell assay for anti-androgenicity of phthalate esters in the rat.
An in vitro assay using the rat Leydig cell line R2C was evaluated for its ability to quantitatively predict inhibition of testosterone synthesis. Results obtained for endocrine active phthalates (MEHP, MBP), and inactive phthalates (MMP and MEP) were highly consistent with in vivo results based on tissue and media concentrations. Statistically significant inhibition of testosterone synthesis (p<0.05, 1-way ANOVA) was observed at 1μM MBP and 3μM MEHP, while MEP and MMP did not affect inhibition of testosterone synthesis until much higher concentrations (≫100μM). Concentrations causing 50% inhibition of testosterone synthesis for MBP and MEHP (3 and 6μM respectively), were similar to in vivo values (3 and 7μM). The R2C assay was used to determine the relative potency of 14 structurally diverse monoesters and oxidative metabolites of MEHP. Monoesters with alkyl chains 4-5 carbons in length had the highest potency for testosterone inhibition, while 0-2 carbon alkyl chains were least potent. Phase I metabolism did not completely inactivate MEHP, underscoring the need for metabolism data when interpreting in vitro pharmacodynamic data. This steroid inhibition assay provides a predictive in vitro alternative to expensive and timeconsuming developmental rat studies for phthalate-induced antiandrogenicity.
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MEP testosterone MBP MEHP MEP MMP testosterone testosterone MBP MEHP MEHP monoesters MEHP Monoesters carbons testosterone carbon phthalate esters steroid testosterone phthalate phthalates MEHP MBP phthalates MMP