Effect of dipentyl phthalate in 3-dimensional in vitro testis co-culture is attenuated by cyclooxygenase-2 inhibition

Exposure to phthalate esters is associated with changes in steroidogenesis, leading to the hypothesis that this is a primary mechanism of phthalate reproductive toxicity. However, some phthalate-induced male reproductive toxicity has been demonstrated in the absence of changes to testosterone production, suggesting additional mechanisms of action. There is evidence that phthalate exposure increases expression of the inflammatory enzyme cyclooxygenase 2 (cox-2). Furthermore, inhibition of cox-2 enhances expression of the steroidogenic acute regulatory protein (StAR), which mediates the rate-limiting step in steroidogenesis. This study hypothesized that phthalate-induced toxicity and testosterone perturbation are mediated in part by cox-2. A 3D in vitro rat testis co-culture to explore the role of cox-2 in phthalate toxicity was employed. Cells were treated with 100 µM dipentyl phthalate (DPP) with and without pre-treatment with the specific cox-2 inhibitor NS-398. Effects were evaluated after 8, 24, and 72 h. DPP exposure significantly increased cox-2 expression at 8 and 24 h (p<0.01) and resulted in significant, dose-dependent cytotoxicity. Pre-treatment with NS-398 significantly reduced the cytotoxicity of DPP at 8 and 24 h (p<0.01). NS-398 also mitigated the effects of DPP on testosterone regulation. Total testosterone concentrations in cell culture media were significantly increased following 8 and 24 hr of DPP exposure (p<0.001) and NS-398 reduced this effect (p<0.05). Simultaneously, DPP significantly decreased StAR protein expression after 8 h (p<0.01) and this effect was significantly attenuated by the presence of NS-398 (p<0.01). These results suggest that the DPP-induced changes in testosterone regulation observed in this experiment are mediated in part by an inflammatory response that is cox-2 dependent.   Key words: dipentyl phthalate, testosterone, cyclooxygenase 2, in vitro toxicology