Qwihee P. Lee

Further studies on the properties of human placental microsomal cytochrome P-450.

Abstract Spectral studies of human placental microsomal cytochromes provided evidence that androstenedione will bind either to two separate cytochromes or to two sites on the same cytochrome. Scatchard and Lineweaver-Burk plots indicated the presence of a binding site with high affinity and low capacity and a second site with a lower affinity but higher capacity. Both sites were discernible in the presence of high concentrations of NADPH, and similar binding constants were obtained. The absorption maximum (450 nm) of the carbon monoxide complex of NADPH-reduced placental cytochrome P-450 could be abolished completely by additions of low concentrations of androstenedione. 19-hydroxyandrostenedione or 19-oxoandrostenedione, but not by high concentrations of 19-norandrostenedione. 19-nortestosterone, pregnenolone or benzo[a]pyrene. Compounds capable of binding significantly to placental microsomal cytochrome P-450 appeared to fall into three categories: C-19 or C-18 steroids, with structures similar to that of androstenedione or 19-norandrostenedione: C-18 steroids with structures very similar to that of β-estradiol; or substances capable of forming ferrihemochromes. A large number of foreign organic compounds which produced type-I binding spectra in rat liver microsomes exhibited either no or very minimal binding to placental cytochrome P-450. Most compounds that exhibited significant binding to placental cytochrome P-450 also were inhibitors of rates of androstenedione aromatizalion (aromatase activity). Important exceptions were carbon monoxide, metyrapone and nicotinamide. Diethylstilbestrol acted as a relatively potent inhibitor of aromatase activity but displayed no discernible binding. Results of the study strongly supported a functional role for placental cytochrome P-450 in the aromatization reaction.

Human embryonic cytochrome p450s: Phenoxazone ethers as probes for expression of functional isoforms during organogenesis

Human embryonic tissues were investigated during the period of organogenesis with a combination of substrate probes, selective inhibitors and immunoprobes in terms of their capacity to express functional P450 isoforms. A series of phenoxazone ethers utilized as substrate probes revealed that human embryonic hepatic, pulmonary, renal, adrenal and cardiac tissues each contained a complement of functional P450 isoforms when analyzed between days 50 and 60 of gestation. Preparations of each of these tissues contained isoforms capable of catalyzing O-demethylation, O-deethylation, O-depentylation and O-debenzylation of the respective phenoxazone ethers. Investigations with chemical inhibitors and inhibitory antibodies as well as comparisons with vector-expressed, human P450 isoforms suggested that isoforms of P450 subfamilies 1A, 2B, 2C or 3A were not major contributors to any of the observed reactions. The P450-dependent reactions studied exhibited several unexpected and unusual characteristics including a preference for NADH over NADPH as the initial electron donor. Results were consistent with the concept that conceptal-specific P450 isoforms participate in the human embryonic O-dealkylation/debenzylation probe reactions investigated.

Cytochrome P450-dependent bioactivation of prodysmorphogens in cultured conceptuses

These investigations were undertaken to determine the extent to which tissues of cultured rat conceptuses contain cytochrome P450 isoforms in sufficient quantities to significantly influence the capacity of certain chemicals to elicit dysmorphogenic effects in vitro. Investigations with highly sensitive probe substrates/inhibitors and with immunologic methods enabled the detection of at least four separate P450 isoforms in tissues of the visceral yolk sac, ectoplacental cone, and embryo proper. One of the isoforms was identified as P450IA1 and was found to be inducible by polycyclic aromatic hydrocarbons in all three tissues. Other isoforms exhibited properties differing from characterized adult rat hepatic isoforms. Each of the isoforms was detectable in conceptuses on gestational days 10, 11, 12, and 14 and was present in the highest concentrations in the visceral yolk sac. Conceptal P450IA1 catalyzed the conversion of dysmorphogenically inactive 2-acetylaminofluorene to 7-hydroxy-2-acetylaminofluorene, a proximate dysmorphogen. Investigations with microinjections suggested that visceral yolk sac hydroxylation was largely responsible for the bioactivation reaction in vitro. The same isoform exhibited no capacity to influence the dysmorphogenic activity of cyclophosphamide. The results demonstrated that tissues of cultured rat conceptuses may contain P450 isoforms in sufficient amounts to markedly influence the dysmorphogenic activity of substrates of the corresponding isoforms.

Differential inhibition of androst-4-en-3,17-dione aromatization by carbon monoxide in the presence of estr-4-en-3,17-dione

The aromatization of androst-4-en-3,17-dione or 17beta hydroxyandrost-4-en-3-one (testosterone) is not inhibited by carbon monoxide under normal incubation conditions, whereas the aromatization of corresponding 19-nor steroids (estr-4-en-3,17-dione and 17beta-hydroxyestr-4-en-3-one) is readily inhibited under the same conditions. A possible explanation was found when it was shown that androst-4-en-3,17-dione and testosterone could displace bound carbon monoxide from human placental microsomal cytochrome P-450. The 19-nor steroids did not displace carbon monoxide, even at very high concentrations. These C-18 compounds appeared to facilitate complex formation and reversed the effects of the C-19 steroids. A mutual antagonism was observed with regard to effects on the formation of the ce titrated. These observations suggested that the aromatization of androst-4-en-3,17-dione should be inhibited by carbon monoxide if sufficient concentrations of the 19-nor steroids were present in reaction flasks. This hypotheses was tested and positive results were obtained, providing strong evidence for the involvement of cytochrome P-450 in normal estrogen biosynthesis.

cAMP-dependent regulation of P450-catalyzed dealkylation in rat conceptal tissues

Exposures of cultured whole rat conceptuses to varying concentrations of dibutyryl cyclic AMP or isobutylmethylxanthine, alone or in combination, resulted in significant increases in rates of cytochrome P450-dependent depentylation of pentoxyphenoxazone in cell-free preparations. Lesser increases in rates of debenzylation of benzyloxyphenoxazone were also observed. In cultured whole conceptuses, basal depentylase and debenzylase activities in the visceral yolk sac were approximately sixfold higher than in the embryo. The ectoplacental cone and decidual tissues exhibited no detectable depentylase activity. Only the visceral yolk sac exhibited increased depentylase activity in response to dibutyryl cyclic AMP and isobutylmethylxanthine. Inhibitory antibodies raised against adult hepatic P450s IIB1, IIC11, and IA1 failed to significantly inhibit the yolk sac depentylase activities of noncultured conceptuses. The results suggested that the conceptal depentylation reaction may be catalyzed by a unique P450 isoform(s) that is not expressed during adult life.

Mechanisms of glucagon-induced increases in rates of cytochrome P450-dependent pentoxyphenoxazone O-depentylation in cultured rat conceptuses.

Inclusions of glucagon (1.0 or 2.0 microM, final concentrations) in the media of cultured whole rat conceptuses resulted in concentration-dependent increases in measured rates of O-depentylation of pentoxyphenoxazone in cell-free preparations of conceptal tissues. Enzymic activities were assayed 24 h after initial exposure of the conceptuses to glucagon on day 10 of gestation. Glucagon elicited increases in tissue levels of cAMP that were parallel to those produced by 3-isobutyl-1-methylxanthine over the same time period. Tissue cAMP levels were maximal after 2 h, rapidly returned to control levels and were also equal to background levels in controls after the 24 h culture period. Dibutyryl cAMP, 3-isobutyl-1-methylxanthine, theophylline, and RO201724, a cAMP-selective phosphodiesterase inhibitor, each produced 75 to 100% increases in O-dealkylase activity. Dibutyryl cGMP and two phosphodiesterase inhibitors, enoximone (cGMP-inhibited) and zaprinast (cGMP-specific), each failed to produce statistically significant increases in O-depentylase activity. The O-depentylase was tentatively identified as a conceptus-specific P450 cytochrome that is synthesized predominantly in tissues of the visceral yolk sac. The results indicated that glucagon may upregulate a unique, xenobiotic-biotransforming P450(s) via a long-term mechanism(s) specifically involving tissue cAMP.

Addition products of uric acid and formaldehyde.

In order to calibrate colorimetric determinations of serum and urine uric acid, standard dilutions are routinely prepared from a stock solution of uric acid in 2% Formalin. We made a similar solution by dissolving 100 mg of uric acid in 100 ml of 1% formaldehyde in water and applied it to a Polyacrylamide column (BIO GEL P-2) monitored spectrophotometrically at 280 nm. Uric acid, dissolved in water or in lithium carbonate solution, characteristically adsorbs to the column matrix and ultimately elutes as a single peak after 33 hours (1). In contrast, the solution of uric acid with formaldehyde unexpectedly resulted in three distinct elution peaks. A small peak was seen with the characteristic slow mobility of free uric acid, but two much larger peaks, designated F2 and F1, were eluted earlier after 19 and 25 hours. This simple chromatographic technique thus demonstrated the presence of two new reaction products and isolated them for further characterization.