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L Milewich

Publications and source records attributed to L Milewich.

At least 37 records · Page 2Linked to original sources
PubMed1990

Steroid sulfatase activity in epidermis of acne-prone and non-acne-prone skin of patients with acne vulgaris.

Abnormal keratinization of hair follicles appears to be intimately associated with acne vulgaris. Whether steroid metabolism in follicular wall keratinocytes of acne-prone skin plays a role in the development and maintenance of acne vulgaris is unknown at this time. The enzymatic hydrolysis of dehydroepiandrosterone sulfate to dehydroepiandrosterone and of estrone sulfate to estrone in cultured epidermal keratinocytes has been demonstrated. Thus, we sought to establish whether steroid sulfatase activity in freshly obtained epidermal tissue (greater than 90% keratinocytes) from acne-prone skin in patients with acne vulgaris was altered when compared with that in epidermal tissue from non-acne-prone skin in the same individuals. We found that there were no differences in the rates of enzymatic hydrolysis of steroid sulfates in epidermis of acne-prone and non-acne-prone skin; however, the rate of estrone sulfate hydrolysis was two to eight times greater than that of dehydroepiandrosterone sulfate in all of the tissues evaluated in this study.

Acne Vulgaris↗
PubMed1990

Cytochrome P-450s as toxicogenic catalysts: the influence of dehydroepiandrosterone.

The cytochrome P-450s catalyze the oxidative transformation of a large number of endogenous and exogenous chemicals in plants, insects, and mammals. One consequence of this type of reaction is the generation of highly reactive electrophilic metabolites that can react with intracellular macromolecules. It has been postulated that the initiation reaction for chemical carcinogenesis and/or cellular toxicity involves the metabolism of xenobiotics by P-450s. The naturally occurring steroid dehydroepiandrosterone (DHEA), when administered as a supplement to the diet of rodents, has been reported to have anti-carcinogenic and other chemoprotective activities. A change in the inventory of liver P-450s occurs during treatment of rodents with DHEA, the most pronounced being that involved in the omega-hydroxylation of medium-chain length fatty acids. In addition, changes in the activities of other liver P-450s, viz., P-450IIB1, P-450IIC11, and P-450IIIA, occur as shown by in vitro experiments to assess the P-450-dependent formation of hydroxylated metabolites of testosterone and androstenedione. The effect of feeding rodents a diet supplemented with DHEA mimics some of the changes seen when animals are treated with hypolipidemic drugs (such as ciprofibrate, and other chemicals which are known to be peroxisome proliferators). Studies comparing the enzymatic functions of the heterologous expressed recombinant forms of P-45017 alpha, responsible for the two step conversions of progesterone and pregnenolone to the C19-steroids, androstenedione, and DHEA, respectively, illustrated the marked differences in enzymatic properties between the human and the rat orthologues. The findings serve to demonstrate that DHEA is an obligatory intermediate for the synthesis of androgens in the human, but not in the rodent. The biochemical changes responsible for the anti-carcinogenic properties of DHEA remain to be identified. The studies presented here suggest that DHEA, when administered as a dietary supplement, functions like a xenobiotic, and that its effects may result from alterations in the inventory of cellular P-450s, thereby influencing the balance of metabolic activities associated with the initiation phase of chemical carcinogenesis and/or toxicity.

Animals↗
PubMed1989

Induction of microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450IVA1 (P-450LA omega) by dehydroepiandrosterone in rats: a possible peroxisomal proliferator.

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Transforming growth factor-beta inhibits prostaglandin production in amnion and A431 cells.

We studied the effect of transforming growth factor-beta (TGF-beta) on prostaglandin E2 (PGE2) production and mitogenesis in human amnion cells and compared the response in amnion cells with that in A431 cells. Both amnion cells and A431 cells respond to epidermal growth factor (EGF) with increased production of PGE2 whereas EGF promotes mitogenesis in amnion cells but not in A431 cells. In amnion cells, TGF-beta was not mitogenic, and did not alter the mitogenic response of cells to EGF. Treatment of amnion cells with TGF-beta did, however, cause a decrease in PGE2 production relative to untreated cells, although EGF stimulated PGE2 production was not attenuated. In A431 cells, TGF-beta acted to decrease PGE2 production relative to untreated cells and to attenuate the stimulation of PGE2 production effected by EGF. The inhibitory action of TGF-beta on PG production in amnion and A431 cells is contrary to the stimulation of PG production in mouse calvaria reported by others and is suggestive that the effect of TGF-beta on prostaglandin production, like its effect on growth, varies between different cell types. Inhibition of PG production by treatment of amnion or A431 cells with mefenamic acid did not alter thymidine incorporation into DNA in response to EGF; similarly, the addition of PGE2 or PGF2 alpha to culture media of amnion or A431 cells had no effect on mitogenesis (in the absence or presence of EGF). Based on these findings, we conclude that PG production and EGF action on proliferation (stimulation in amnion cells; inhibition in A431 cells) are dissociated.

Amnion↗
PubMed1989

Cachectin/tumor necrosis factor-alpha formation in human decidua. Potential role of cytokines in infection-induced preterm labor.

This study was conducted as part of an investigation to evaluate the hypothesis that bacterial toxins (LPS or lipoteichoic acid), acting on macrophage-like uterine decidua to cause increased formation of cytokines, may be involved in the pathogenesis of infection-associated preterm labor. We found that cachectin/tumor necrosis factor-alpha (TNF-alpha) was synthesized and secreted into the culture medium by human decidual cells and explants in response to treatment with LPS. LPS treatment also caused an increase in PGF2 alpha production by decidual cells and explants. In amnion cells in monolayer culture, TNF-alpha stimulated PGE2 formation, and TNF-alpha was cytostatic (inhibited [3H]thymidine incorporation into DNA) but not cytolytic in amnion cells. TNF-alpha was not detectable (less than 0.34 ng/ml) in the amniotic fluid of normal pregnancies at midtrimester or at term before or after the onset of labor (n = 44); but TNF-alpha was present at concentrations between 2.8 and 22.3 ng/ml in amniotic fluids of 4 of 20 pregnancies with intact membranes complicated by preterm labor (less than 34 wk gestational age). LPS was present in 10 of the 20 amniotic fluids of preterm labor pregnancies, including all four in which TNF-alpha was present. Bacteria were identified in only one of the four LPS-positive, TNF-alpha-positive fluids. Cytokine formation in macrophage-like decidua may serve a fundamental role in the pathogenesis of preterm labor, including increased prostaglandin formation and premature rupture of the membranes.

Amniotic Fluid↗
PubMed1989

Activity of 17 beta-hydroxysteroid oxidoreductase in tissues of the human fetus.

The interconversion of oestrone and oestradiol, androstenedione and testosterone, and dehydroepiandrosterone and 5-androstene-3 beta,17 beta-diol in mammalian tissues is catalysed by 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR). To identify tissue sites of 17 beta-HSOR activity in the human fetus, microsomal fractions from 15 different fetal tissues obtained from first and second trimester pregnancies were used for evaluation of enzymatic activity by use of [17 alpha-3H] oestradiol as the substrate and NADP+ as the co-factor. With these reagents, the enzyme-catalysed reaction led to the production of both non-radiolabelled oestrone and NADP3H in equimolar amounts; the radioactivity associated with NADP3H was used to quantify 17 beta-HSOR activity. Activity of 17 beta-HSOR was present in microsomes of all the tissues evaluated. The specific activity of the enzyme was highest in liver and placental microsomes. The interconversion of oestradiol and oestrone in microsomal fractions of nine different fetal tissues was studied by the use of substrates labelled with tritium at stable nuclear positions ([6,7-3H]oestradiol and [6,7-3H]oestrone). The products, [3H]oestrone and [3H]oestradiol, were quantified by the use of established techniques; other metabolites formed in these incubations were not identified. The reductive pathway of metabolism (oestrone to oestradiol) appeared to be favoured in microsomal fractions prepared from placenta, fetal zone of the adrenal gland and, possibly, lung. The oxidative pathway (oestradiol to oestrone) appeared to be favoured in microsomes prepared from liver, intestine, stomach, kidney, brain and heart. 17 beta-HSOR activity in fetal liver also was assessed by the use of fresh and frozen-thawed tissue, homogenate, subcellular fractions, and, also, in primary hepatocytes maintained in culture; the specific activity of the enzyme was highest in the microsomal fraction of liver tissue and 17 beta-HSOR activity in liver microsomes was linear with time of incubation up to 1 h. In hepatocytes, the enzymatic activity was linear with time of incubation up to 2 h and with cell number up to 2.5 x 10(5) cells/ml; the apparent Michaelis constant of hepatocyte 17 beta-HSOR for oestradiol was 11 mumol/l. The specific activity of 17 beta-HSOR did not change after pretreatment of hepatocytes for 24 h with insulin, glucagon or dexamethasone.

17-Hydroxysteroid Dehydrogenases↗
PubMed1988

Prostacyclin biosynthesis by cultured human myometrial smooth muscle cells: dependency on arachidonic or linoleic acid in the culture medium.

Myometrial smooth muscle cells in culture were incubated for 18 hours in medium that contained serum (10%); under these conditions, there was a six to 26-fold increase in the amount of 6-keto-prostaglandin F1 alpha that accumulated in the medium (i.e., prostacyclin production) compared with that present after incubation in serum-free medium. In serum-free and serum-containing media, treatment of these cells with dexamethasone (10(-8) mol/L) or cortisol (10(-7) mol/L) suppressed the biosynthesis of prostacyclin by approximately 80% and 64%, respectively. Arachidonic acid (bound to fatty acid-free human serum albumin) added to serum-free medium caused a concentration-dependent increase in the production of prostacyclin by myometrial cells. Arachidonic acid was maximally effective at a concentration of 10(-5) mol/L and caused a five- to 28-fold increase in the biosynthesis and secretion of prostacyclin. Linoleic acid (bound to albumin) in serum-free medium also caused a concentration-dependent increase in the production of prostacyclin; however, the amount of prostacyclin produced in the presence of linoleic acid was lower than that produced in the presence of an equimolar concentration of arachidonic acid. In the presence of arachidonic (10(-5) mol/L) or linoleic acids (10(-4) mol/L) in serum-free medium, the addition of dexamethasone (10(-8) mol/L) or cortisol (10(-7) mol/L) suppressed but did not inhibit completely prostacyclin production. These findings are indicative that arachidonic and linoleic acids in the culture medium support prostacyclin biosynthesis by human myometrial smooth muscle cells. The inhibition of prostacyclin production by glucocorticosteroids in the absence or presence of extracellular arachidonic (or linoleic) acid may be caused by inhibition of phospholipase A2 activity.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Epidermal growth factor, vanadate, and 12-O-tetradecanoylphorbol-13-acetate inhibit growth and stimulate prostaglandin E2 production in A431 cells.

The cell line designated as A431 is characterized by an extraordinary capacity for binding of epidermal growth factor (EGF); but paradoxically, these cells also are characterized by failure of replication in response to treatment with EGF and other mitogens. In this study, we took advantage of this unique response of A431 cells to EGF and other mitogenic agents. Specifically, we investigated the dependency of mitogen-stimulated prostaglandin production on mitogenesis. The effects of treatment of A431 cells with epidermal growth factor and two other mitogenic agents, viz., sodium orthovanadate and 12-O-tetradecanoylphorbol-13-acetate (TPA), on DNA synthesis and prostaglandin production were evaluated. The rate of prostaglandin production in A431 cells that were treated with each of these agents increased in a manner that was dependent upon the duration of treatment and the concentration of the agent tested. Thus, by use of A431 cells, we find that the effects of 'mitogenic' agents on cell replication and prostaglandin production are clearly dissociable.

Cell Line↗
PubMed1988

Steroid metabolism by epidermal keratinocytes.

The metabolism of various radiolabeled steroids by cultured human epidermal keratinocytes was studied in an attempt to identify the steroid-metabolizing enzymes present in these cells. Sulfatase activity was demonstrated in keratinocytes with either E1S or DS as substrates. The products of sulfatase action were E1 and DHEA, respectively. The specific activity of the enzyme was approximately 5- to 14-fold greater with E1S as the substrate compared with DS, and the rates of hydrolysis were linear with incubation time up to 3 h. The metabolism of DHEA by the keratinocyte 17 beta-HSOR-catalyzed reaction resulted in the predominant formation of 5-androstene-3 beta,17 beta-diol. The rate of formation of 5-androstene-3 beta,17 beta-diol was linear with time of incubation up to 18 h, and the specific activity of 17 beta-HSOR, with DHEA as the substrate, was greater in keratinocytes maintained in culture for 4 weeks compared with keratinocytes kept in culture for 1 week. Androstenedione was a minor product of DHEA metabolism. The metabolism of DHT by epidermal keratinocytes resulted in the formation of 5 alpha-androstanedione, 5 alpha-androstane-3 alpha,17 beta-diol, 5 alpha-androstane-3 beta,17 beta-diol, androsterone, and isoandrosterone: the rates of formation of 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol were linear with incubation time up to 24 h, and the specific activities of 3 alpha-HSOR and 3 beta-HSOR did not appear to change with keratinocyte time in culture up to 3 weeks. The metabolism of DOC by epidermal keratinocytes resulted in 5 alpha-dihydrodeoxycorticosterone production: the rate of formation of this metabolite was linear with incubation time up to 4 h. The metabolism of E1 by epidermal keratinocytes yielded E2, and that of E2 resulted in the formation of E1. The rate of E1 formation from E2, was approximately 10-fold greater than the rate of formation of E2 from E1; these rates were linear with incubation time up to 4 h. Epidermal keratinocytes maintained in culture did not metabolize androstenedione to either E1 or E2, and pregnenolone was not metabolized by these cells. This study serves to ascertain that epidermal keratinocytes express steroid 5 alpha-reductase, 17 beta-HSOR, 3 beta-HSOR, 3 alpha-HSOR, 3 beta-hydroxysteroid oxidoreductase-delta 5----4-isomerase, and sulfatase activities.

Arylsulfatases↗
PubMed1987

Steroid 5 alpha-reductase activity in endothelial cells from human umbilical cord vessels.

The metabolism of radiolabeled progesterone and androstenedione was evaluated in endothelial cells from human umbilical cord vein and arteries maintained in culture. The predominant metabolite of progesterone was 5 alpha-pregnane-3,20-dione and that of androstenedione was 5 alpha-androstane-3,17-dione. Thus, the major pathway of progesterone and androstenedione metabolism within these cells is via steroid 5 alpha-reductase. The rate of formation of 5 alpha-pregnane-3,20-dione from progesterone by venous endothelial cells was linear with incubation time up to 4 h and with cell number up to 1.6 X 10(6) cells/ml. The apparent Km of 5 alpha-reductase for progesterone was 0.4 microM; and, the Vmax was 55 pmol 5 alpha-pregnane-3,20-dione formed/mg protein X h. The rate of 5 alpha-androstane-3,17-dione formation from androstenedione also was linear with incubation time up to 4 h. In addition to 5 alpha-androstane-3,17-dione, the metabolism of androstenedione by either venous or arterial cells resulted in the formation of various minor metabolites, including testosterone and 5 alpha-reduced steroids, viz. 5 alpha-dihydrotestosterone, androsterone, isoandrosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, and 5 alpha-androstane-3 beta, 17 beta-diol. Estrogens (i.e. estradiol-17 beta and estrone) were not detected as products of androstenedione metabolism. The formation of these metabolites are indicative that the steroid-metabolizing enzymes present in endothelial cells are: 5 alpha-reductase, 17 beta-hydroxysteroid oxidoreductase, 3 alpha-hydroxysteroid oxidoreductase, and 3 beta-hydroxysteroid oxidoreductase.

5-alpha-Dihydroprogesterone↗
PubMed1987

In situ steroid sulfatase activity in human epithelial carcinoma cells of vaginal, ovarian, and endometrial origin.

The enzymatic hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate to estrone and dehydroisoandrosterone, respectively, was studied in cells that were derived from four different malignant tumors of the lower reproductive tract of women, viz. a squamous cell vaginal carcinoma, an ovarian carcinoma, and two endometrial adenocarcinomas. These cells had the capacity to hydrolyze both steroid sulfoconjugates. Estrone sulfate was more efficient as a substrate than dehydroepiandrosterone sulfate, since the amount of product formed from estrone sulfate was approximately 3-fold greater than that formed from dehydroepiandrosterone sulfate. Some kinetic parameters of steroid sulfatase were determined in the four cell types and were found to be very similar, as were the rates of hydrolysis. Sulfatase activity was linear with incubation time for at least 2 h and with cell number up to 3.2 X 10(6) cells/mL. The apparent pH optimum of steroid sulfatase, determined by the use of cell sonicates and estrone sulfate as the substrate, was between 6.0 and 7.5. The apparent Km values of steroid sulfatase for estrone sulfate in both squamous vaginal carcinoma cells and ovarian carcinoma cells were both 5 microM, and those for dehydroepiandrosterone sulfate in squamous vaginal carcinoma cells and endometrial adenocarcinoma cells were 6 and 4 microM, respectively. The optimal temperature of steroid sulfatase in squamous vaginal carcinoma cells was 50 C; at this temperature, enzymatic activity was more than twice that at 37 C. The steroid sulfatase pathway that is operative in carcinoma cells in vitro to produce free steroids from steroid sulfate precursors also may serve to produce free steroids in vaginal, endometrial, and ovarian carcinomas in vivo and, perhaps, maintain and stimulate tumor growth.

Adenocarcinoma↗
PubMed1986

Metabolism of dehydroisoandrosterone and androstenedione by human A-549 alveolar type II epithelial-like cells.

The A-549 cell line was initiated from an explant of human lung carcinoma tissue. The biochemical characteristics of these cells are similar to those of normal alveolar type II epithelial cells. To gain some insight into the steroid-metabolizing capabilities of A-549 cells, the metabolism of tritium-labeled dehydroisoandrosterone and androstenedione by these cells was studied. The metabolism of dehydroisoandrosterone led to the exclusive formation of 5-androstene-3 beta,17 beta-diol. The major product of androstenedione metabolism was testosterone; and, 5 alpha-reduced steroids also were formed, viz. 5 alpha-androstane-3,17-dione, androsterone, isoandrosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol. Estrogens, viz., estrone and estradiol-17 beta, were not products of androstenedione metabolism by A-549 cells. The rates of metabolite formation from either dehydroisoandrosterone or androstenedione were linear as a function of incubation time up to 3 h, and with cell number up to 1 X 10(6) cells/ml. The apparent Km of 17 beta-hydroxysteroid oxidoreductase for dehydroisoandrosterone was 11 microM, and that for androstenedione was 13 microM. The predominant formation of 5-androstene-3 beta,17 beta-diol from dehydroisoandrosterone, and testosterone from androstenedione is a likely indication that the principal C19-steroid-metabolizing enzyme in A-549 cells is 17 beta-hydroxysteroid oxidoreductase; the other steroid-metabolizing enzymes expressed in these cells are 5 alpha-reductase, 3 beta-hydroxysteroid oxidoreductase and 3 alpha-hydroxysteroid oxidoreductase. The findings of this study demonstrate that A-549 cells express steroid-metabolizing enzymatic activities that are qualitatively similar to those found in other human pneumonocytes and human lung tissue, except for 3 beta-hydroxysteroid oxidoreductase-5----4-isomerase activity, which is not expressed in these cells with dehydroisoandrosterone as the substrate.

Androstenedione↗
PubMed1986

Androstenedione metabolism in human lung fibroblasts.

Human lung fibroblasts in culture metabolized [3H]androstenedione to a number of different compounds, including testosterone, 5 alpha-androstanedione, androsterone, 5 alpha-dihydrotestosterone, isoandrosterone, and 5 alpha-androstane-3 alpha,-17 beta-diol. The major products were 5 alpha-androstanedione and testosterone. Estrone, estradiol-17 beta and 5 beta-reduced steroids were not formed. The production rates of testosterone and 5 alpha-androstanedione from [3H]androstenedione by lung fibroblasts were studied both as a function of incubation time and substrate concentration. The rates of formation of testosterone and 5 alpha-androstanedione remained linear with time up to 4 h. The apparent Km of human lung fibroblast 5 alpha-reductase was 1 microM, and that of 17 beta-hydroxysteroid oxidoreductase was 11 microM. The findings of this study suggest that mesenchyma may contribute to the metabolism of androstenedione in human lung tissue.

Androstenedione↗
PubMed1986

Epidermal keratinocytes: a source of 5 alpha-dihydrotestosterone production in human skin.

The major products of testosterone, androstenedione, and progesterone metabolism by human epidermal keratinocytes are 5 alpha-reduced steroids, viz. 5 alpha-dihydrotestosterone, 5 alpha-androstanedione, and 5 alpha-dihydroprogesterone, respectively. The rates of metabolite formation by these cells were linear with incubation time up to 3 h. The apparent Km of keratinocyte 5 alpha-reductase was 1.3 microM for androstenedione and 1.5 microM for progesterone. 5 alpha-Reductase activity was found only in particulate subcellular fractions of a homogenate of epidermal keratinocytes when assayed with tritium-labeled progesterone as the substrate and NADPH as the cofactor. In addition to 5 alpha-reductase activity, other enzymatic activities found in epidermal keratinocytes were 17 beta-hydroxysteroid oxidoreductase and 3 beta-hydroxysteroid oxidoreductase. These enzymes were expressed in the formation of androstenedione from testosterone, testosterone from androstenedione, isoandrosterone from androstenedione, and 3 beta-hydroxy-5 alpha-pregnan-20-one from progesterone. The apparent Km of 17 beta-hydroxysteroid oxidoreductase for androstenedione in epidermal keratinocytes was 10 microM. When measured at weekly intervals, the rates of product formation from testosterone, androstenedione, or progesterone by cultured epidermal keratinocytes increased several-fold with advancing time in culture up to 3 weeks. The results of these studies suggest that epidermal keratinocytes are a major site of synthesis of biologically potent androgens in human skin, viz. testosterone from androstenedione and 5 alpha-dihydrotestosterone from testosterone. Skin is a target organ for 5 alpha-dihydrotestosterone action, and thus, the local formation of 5 alpha-dihydrotestosterone may play an important role in the regulation of proliferation and differentiation of keratinocytes.

Adult↗
PubMed1986

Human fetal liver estrogen 16 alpha-hydroxylase: precursor specificity, kinetic parameters, and in vitro regulation.

The properties of human fetal liver (HFL) estrogen 16 alpha-hydroxylase (16 alpha-OHase) were studied in microsomal preparations and hepatocytes maintained in culture. A specific assay was developed for the determination of estrogen 16 alpha-OHase activity based on the enzymatic release of tritium from the C-16 alpha position of stereospecifically labeled 16 alpha-3H-labeled C18-steroids, viz. 17 beta-[16 alpha-3H]estradiol, 17 beta-[16 alpha-3H]estradiol 3-sulfate, [16 alpha-3H]estrone, and [16 alpha-3H]estrone sulfate. The percentage of tritium at the C-16 alpha position of 17 beta-[16 alpha-3H]estradiol was 92.5%. There was no kinetic isotope effect in the 16 alpha-hydroxylation of 17 beta-[16 alpha-3H]estradiol. HFL hepatocyte and microsomal 16 alpha-OHase activity was linear with incubation time for at least 2 h, with a hepatocyte number up to 6.7 X 10(6) cells/ml and a microsomal protein concentration up to 1 mg/ml. The apparent Km of 16 alpha-OHase for estrone sulfate (E1S) was greater than that for either 17 beta-estradiol (E2) or estrone (E1; 2.9-6.4 microM vs. 0.70-0.84 microM). The maximum velocity of HFL 16 alpha-OHase also was greater with E1S or 17 beta-estradiol 3-sulfate than with either E1 or E2, and E1S was the most efficient substrate. The apparent temperature optimum for the microsomal enzyme was 37 C, and the apparent pH optimum was 7.0. 16 alpha-Hydroxylation of E1S by HFL microsomes was inhibited noncompetitively by E1. The biosynthesis of estriol from E2 by fetal liver microsomes does not require an intermediate oxidation step of E2 to E1 as appears to be the case in vivo in the human adult; this was demonstrated by the formation of [17 alpha-3H]estriol from [17 alpha-3H] E2 in incubations with HFL microsomes. A number of growth factors, hormones, and xenobiotics were preincubated with hepatocytes for 24 or 72 h to test for stimulation/inhibition of 16 alpha-OHase activity. 16 alpha-OHase activity was stimulated by dexamethasone, forskolin, (Bu)2cAMP, cholera toxin, 1,2-benzanthracene, and phenobarbital. Diethylstilbestrol, E2, and progesterone did not alter hepatocyte 16 alpha-OHase activity, except when very high concentrations of E2 and progesterone were used, when they became inhibitory; other hormones and growth factors did not alter the basal levels of the enzyme.

Anencephaly↗
PubMed1986

Estrogen 16 alpha-hydroxylase activity in human fetal tissues.

Estrogen 16 alpha-hydroxylase activity was measured in microsomes prepared from fetal tissues of first and second trimester human abortuses using [16 alpha-3H]estrone sulfate as substrate and NADPH as cofactor. Estrogen 16 alpha-hydroxylase activity was demonstrable in 13 of 14 fetal tissues examined in this study, viz. liver, adrenal fetal zone, adrenal neocortex, lung, kidney, intestine, heart, brain, skin, testis, spleen, pancreas, and stomach, and was either negligible or absent in placental tissue. The highest specific activity of the microsomal enzyme [pico-moles of product(s) formed per mg protein/h] was found in liver (mean +/- SEM, 338 +/- 62), and the next highest was found in the fetal zone of the adrenal cortex (70 +/- 20). The specific activities of estrogen 16 alpha-hydroxylase in adrenal neocortex, brain, skin, and testis were similar (25-53 pmol/mg protein X h) as were those in lung, kidney, intestine, heart, spleen and stomach (23-36 pmol/mg protein X h). The specific activity of the enzyme in the pancreas was 12 pmol/mg protein X h; the lowest specific activity, however, was in placental microsomes (0.2 +/- 0.1 pmol/mg protein X h).

Adrenal Glands↗
PubMed1986

Lecithin-sphingomyelin ratios in amniotic fluid of pregnancies with an anencephalic fetus.

As many investigators have shown that surfactant production in the developing human lung is subject to multihormonal regulation, the present authors determined the lecithin-sphingomyelin (L/S) ratio in amniotic fluid of pregnancies with an anencephalic fetus, in which there was known to be aberrant production of fetal pituitary, adrenal, and consequently, placental hormones. The L/S ratio in amniotic fluid from seven of eight pregnancies with an anencephalic fetus was substantially lower than that in amniotic fluids of pregnancies with a normal fetus at the same stage of gestation. The L/S ratio in amniotic fluid of an anencephalic fetus of a twin pregnancy (monochorionic diamniotic) at 34 weeks' gestation was low; the L/S ratio of the amniotic fluid of the normal fetus was high. These data are supportive of the view that fetal lung maturation is dependent, in part, upon normal function of the fetal pituitary and adrenal.

Amniotic Fluid↗