Conversion of pregnenolone to DHEA by human 17 alpha-hydroxylase/17,20-lyase (P450c17) - Evidence that DHEA is produced from the released intermediate, 17 alpha-hydroxypregnenolone

Most previous studies using reconstituted systems and fast kinetics suggest that the conversion of pregnenolone to dehydroepiandrosterone (DHEA; the p recursor of androgen and estrogen biosynthesis) by P450c17 does not require the release of the intermediate 17 alpha-OHPreg (a precursor of cortisol b iosynthesis). With such a mechanism, it is difficult to conceive how high a mounts of DHEA may be produced in some cells or tissues, such as the testis and cells from the adrenal reticularis, while in other tissues such as the fasciculata zone, high levels of 17 alpha-OHPreg are synthesized. In this report, we address this matter using intact transfected cells, which better reflect the actual cellular conditions. Furthermore, by using transfected cells, we can conveniently analyze human enzymes, as we are not restricted by the availability of human tissues as in the case of methods using purifi ed or partially purified enzymes. Using intact HEK-293 cells transfected wi th human P450c17 in culture, we showed, in a time course study of the trans formation of pregnenolone, that there is an accumulation of 17 alpha-OHPreg , and that, subsequently, the accumulated 17 alpha-OHPreg decreases with a concomitant increase in DHEA production. The DHEA/17 alpha-OHPreg ratio cha nges from 0.1 :1 after 1 h incubation to 50 : 1 after 20 h. This result str ongly suggests that the transformation of Preg to DHEA proceeds through two steps in which DHEA is produced from the released intermediate 17 alpha-OH Preg. We also show that high levels of substrate vs. enzyme concentration w ill lead to high hydroxylase activity whereas the reverse will increase the lyase activity. The result is in good agreement with recent observations s uggesting that surrounding enzymes and steroids could modulate the lyase ac tivity. Cotransfection of vectors expressing cytochrome b5 and NADPH cytoch rome P450 reductase indicates that both are required for an optimum product ion of DHEA.