Inhibition of steroid production in Leydig cells by non-steroidal anti-inflammatory and related compounds: evidence for the involvement of lipoxygenase products in steroidogenesis

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Abstract
The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumor Leydig cells was investigated. In the presence of nordihydroguaiaretic acid {NDGA; 4,4''-(2,3-dimethylbutan-1,4-diyl)bis[1,2-benzenediol]}, 5,8,11,14-eicosatetraynoic acid (ETYA), BW755C {3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride} and benoxaprofen [Opren; 2-(2-p-chlorophenyl-benzoxazol-5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 .mu.M for NDGA, ETYA, BW755C and benoxaprofen were obtained, respectively). BW755C and benoxaprofen had no significant effect on LH-stimulated cAMP production except at the highest concentrations examined (330 and 380 .mu.M, respectively), whereas NDGA and ETYA inhibited LH-stimulated cAMP production in a dose-dependent manner (ID50 7.0 and 22 .mu.M, respectively). NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cAMP-stimulated testosterone and prenenolone production. The metabolism of exogenous (22R)-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and (22R)-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]-arachidonic acid by purified rat tumor Leydig cells was investigated indicate that products are formed by tumor Leydig cells that have similar mobilities in a TLC system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW755C and benoxaprofen but not by aspirin and indomethacin. Inhibition of lipoxygenase activity but not cyclo-oxygenase activity caused an inhibition of LH- and dibutyryl cAMP-stimulated steroid production. A stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis is suggested. The site of this stimulation is apparently distal to the production of cAMP and before the side chain cleavage of cholesterol.