Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process.

Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.     

Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

Hormonal effects on the regulation of hepatic heme biosynthesis

Summary Drug-induced porphyrin accumulation occurs in chick embryo liver cells maintained in serum-free Waymouth MD 705/1 medium. Addition of insulin and thyroxine to the medium results in a marked enhancement of porphyrin accumulation. The addition of hydrocortisone results in a further enhancement of porphyrin accumulation.Several agents which are reported to increase intracellular adensosine 3:5-monophosphate (cAMP) levels, viz. glucagon, sodium fluoride, cAMP or its dibutyryl derivative, 3-isobutyl-1-methylxanthine and papaverine enhanced drug-induced porphyrin biosynthesis. On the other hand, agents which are reported to decrease intra-cellular cAMP levels, viz. alloxan and imidazole, diminished drug-induced porphyrin accumulation. cAMP appears to enhance, but not to function as a second messenger in drug-induced porphyrin biosynthesis.Drug-induced porphyrin accumulation in chick embryo liver cells depend upon the insulin to glucagon ratio. A low level of porphyrin accumulation occurs at insulin to glucagon ratios similar to those found following glucose administration in vivo, suggesting a possible explanation for the therapeutic effect of glucose in hepatic porphyria.The 5H(A:B trans) and 5H(A:B cis) steroids are equipotent in inducing -aminolevulinic acid synthetase and porphyrin accumulation in chick embryo liver cells maintained in serum-free culture medium. Thus, there is no specific steric requirement for porphyrin-inducing activity in steroids.This work was supported by the Medical Research Council of Canada.     

Inhibition of steroid-mediated induction of δ-aminolevulinic acid synthase by 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A)

The inhibition of the steroid-mediated induction of δ-aminolevulinate synthase, the rate-limiting enzyme in hepatic porphyrin-heme biosynthesis, by 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A) as studied in cultured chick embryo liver cells. The formation of porphyrins in response to cyproterone, a synthetic steroid, was inhibited in a time-dependent manner by SKF 525-A, an inhibitor of several drug metabolizing enzyme systems. This action is a result of an inhibitory effect of SKF 525-A on the cyproterone-mediated induction of δ-aminolevulinate synthase; SKF 525-A laso inhibited the induction of the enzyme by the naturally occurring 5β-H steroids, etiocholanolone and pregnanolone. Employing [³H]etiocholanolone, we provide evidence that this inhibition is not associated with either decreased uptake or an altered metabolism of the steroid. Moreover, approx. 4–6-fold more radioactivity was associated with [³H]etiocholanolone-treated cells cultured in the presence of SKF 525-A. Alternative mechanisms for the induction of δ-aminolevulinate synthase by steroids are proposed which do not require the interaction of steroid-receptor complex with the genome.     

The effect of beta-adrenergic blocking agents on experimental porphyria induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in vivo and in vitro.

The effect of DL-propranolol on 3',5'-diethoxycarbonyl-1,4-dihydrocollidine-induced experimental porphyria was studied. DL-Propranolol, a beta-adrenergic blocking agent with non-specific membrane effects, partially inhibited 3',5'-diethoxycarbonyl-1,4-dihydrocollidine-induced delta-aminolevulinate synthetase activity both in rats and in chick embryo liver cells in culture. In rats, DL-propranolol decreased urinary delta-aminolevulinate and porphobilinogen but no change occurred in the 24-h urinary excretion of total porphyrins and in the concentration of porphyrins in the liver. In cultured chick embryo liver cells treated with 3',5'-diethoxycarbonyl-1,4-dihydrocollidine, DL-propranolol decreased accumulation of porphyrins in the medium. D-Propranolol, oxprenolol and quinidine acted like DL-propranolol in chick embryo liver cells in culture treated with 3',5'-diethoxycarbonyl-1,4-dihydrocollidine. Pindolol, practolol and lidocaine had no effect. Phenobarbitone had a synergistic effect on the induction of delta-aminolevulinate synthetase by 3',5'-diethoxycarbonyl-1,4-dihydrocollidine in cultures of chick embryo liver cells. This induction was partially inhibited by propranolol. However, the increased accumulation of porphyrins in the medium caused by 3',5'-diethoxycarbonyl-1,4-dihydrocollidine was inhibited by the addition of phenobarbitone. This inhibited induction was further decreased by propranolol. Most of our results indicate that the drugs tested act mainly by their effects on membranes.     

Effects of antihypertensive drugs on hepatic heme biosynthesis,and evaluation of ferrochelatase inhibitors to simplify testing of drugs for heme pathway induction

Effects of a series of antihypertensive drugs on the activity of δ-aminolevulinate synthase and on the formation of porphyrins and cytochrome P-450 were examined in the 18-day-old chick embryo liver in ovo. Hydralazine, pargyline, phenoxybenzamine, clonidine, and spironolactone were found to induce δ-aminolevulinate synthase in this system. These drugs therfore have the potential to precipitate clinical expression in human hereditary hepatic porphyrias and should be avoided or used with caution in patients with these disorders. Differential effects of these and other drugs were observed in the avian liver, in that δ-aminolevulinate synthase was more commonly induced thatn were porphyrins and cytochrome -450; the synthase was usually highest 6–12 h after injection, whereas porphyrins and cytochrome P-450 were highest at 24 h. Furthermore marked porphyrin accumulation was not seen with many drugs that induce σ-aminolevulinate synthase and cytochrome P-450 but was more characteristic of compounds that reduced the metabolism of protoporphyrin to heme, such as 1,4-dihydro-3,5-dicarbethoxycollidne (DDC) and high dose of hydralazine. A sensitive and convenient method to test for capacity to induce heme biosynthesis was adapted for use in the chick embryo liver. This employed a relatively small “priming” dose (0.25 mg) of DDC given with a drug being tested and a fluorometric assay of porphyrins in a liver homogenate obtained at 24 h. This simple method should facilitate screening for those drugs which induce the synthesis of δ-aminolevulinate synthase and/or cytochrome P-450 and are potentially dangerous to patients with hereditary hepatic porphyria.     

The effect of beta-adrenergic blocking agents on experimental porphyria induced by 3,5-diethoxycarbonyl-1,4-dihydrocollindine (DDC) in vivo and in vitro

The effect of DL-propranolol on 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine-induced experimental porphyria was studied.dl-Propranolol, a beta-adrenergic blocking agent with non-specific membrane effects, partially inhibited 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine-induced delta-aminolevulinate synthetase activity both in rats and in chick embryo liver cells in culture.In rats, DL-propranolol decreased urinary delta-aminolevulinate and porphobilinogen but no change occurred in the 24-h urinary excretion of total porphyrins and in the concentration of porphyrins in the liver. In cultured chick embryo liver cells treated with 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine, DL-propranolol decreased accumulation of porphyrins in the medium.d-Propranolol, oxprenolol and quinidine acted like dl-propranolol in chick embryo liver cells in culture treated with 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine. Pindolol, practolol and lidocaine had no effect.Phenobarbitone had a synergistic effect on the induction of delta-aminolevulinate synthetase by 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine in cultures of chick embryo liver cells. This induction was partially inhibited by propranolol. However, the increased accumulation of porphyrins in the medium caused by 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine was inhibited by the addition of phenobarbitone. This inhibited induction was further decreased by propranolol.Most of our results indicate that the drugs tested act mainly by their effects on membranes.     

Acidic metabolites. VI. 20 alpha-isosteroids as intermediates in 20 alpha-dihydrosteroid formation

We have previously shown that human subjects metabolize the 20 beta-epimer of isocortisol (11 beta, 17,20 beta-trihydroxy-3-oxo-pregn-4-en-21-al) to both 20 alpha- and 20 beta-hydroxy steroid end products. In this paper we describe the synthesis of tritium labeled 20 alpha-epimers of isocortisol and isoTHF (3 alpha, 11 beta, 17,20 alpha-tetrahydroxy-5 beta-pregnan-21-al) and their metabolic fate in humans. Both steroids yielded 20 alpha-hydroxy urinary neutral end-products (cortols and cortolones) and no 20 beta-hydroxy epimers. Regeneration of 17-ketols from aldols occurred to a small extent with isoTHF, but not with isocortisol. Isocortisol and isoTHF yielded less cortoic acids than did the corresponding ketols. The results provide further evidence that in man the stereochemistry at C-20 of the end-products of corticosteroid metabolism is determined by the configuration of the aldol at C-20 prior to subsequent metabolic events.     

The altered specificity of cortisone reductase with certain retroandrostan-3-one substrates.

The retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione were good substrates for cortisone reductase in the presence of NADH, and the products corresponded to the respective 3beta-hydroxy compounds, in which the 3beta-hydroxyl group is axial and the absolute configuration is 3S. The analogous natural steroids 17beta-hydroxy-5beta,9alpha,10beta-androstan-3-one and 5beta,9alpha,10beta-androstane-3,17-dione were very poor substrates, and gave the corresponding 3alpha(equatorial,3R)-hydroxy compounds, and, in the latter case, also an appreciable amount of 3beta(axial, 3S)-hydroxy-5beta,9alpha,10beta-androstan-17-one. The natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione were better substrates than the retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one, but were not such good substrates as the retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione. Unlike these retro steroid 5beta,9beta,10alpha-androstan-3-ones, the natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione gave the corresponding 3alpha(axial,3R)-hydroxy compounds. The retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one was not a good substrate, and the product of reaction corresponded to the 3alpha(axial,3R)-hydroxy compound. The nature of substrate recognition by this enzyme is discussed in the light of these structure-activity relationships.     

Insulin inhibition of tyrosine aminotransferase induction by dexamethasone is species-related

Dexamethasone induces TAT in the embryonic chick liver, but this effect is not blocked by prior administration of insulin at a dose sufficient to increase the hepatic protein/DNA ratio. Similarly, insulin does not block dexamethasone induction of TAT in chick embryo hepatocytes in vitro. In contrast, insulin reduces TAT induction by dexamethasone in fetal rat hepatocytes by 35-40%. No significant differences in insulin binding were noted between chick and rat hepatocytes.     

Influence of the strain of rats on the induction of hexachlorobenzene induced porphyria

1. The present work undertakes a comparative study on the hexachlorobenzene (HCB) porphyria induction in female rats of Wistar and CHBBTHOM strains. The purpose was to characterize the CHBBTHOM strain with respect to the haem metabolic pathway, its regulatory mechanisms and its response to foreign drugs. 2. After 7 weeks of treatment it was observed that the hepatic porphyrins increased 140 times, ALA-synthase 4 times and PCL was 73% inhibited in the Wistar strain. 3. On the other hand the animals of CHBBTHOM strain showed lesser alteration on these parameters; hepatic porphyrins increased only 3-fold, ALA-synthase 1.7-fold and PLC was only 22% inhibited. 4. Total iron liver content was nearly equal in both strains of rats. 5. The results obtained would indicate that the lower susceptibility of the CHBBTHOM strain to acquire porphyria does not seem to be due to either: (1) congenital alterations of any parameters of the haem metabolic pathway, since the behaviour of normal animals from both strains was similar; or (2) a lower hepatic iron content in such animals. 6. These findings would suggest that the differential response to HCB to this strain would be looked for in another metabolic pathway, such as that involved in the metabolization process of the toxic.     

Modulating effects of beta-naphthoflavone on the induction of SCEs by model compounds with emphasis on benzo[a]pyrene

The inducing capability of the synthetic flavonol beta-naphthoflavone (beta-NF) on cytochrome P-450 content was studied in primary chick embryo hepatocytes. In addition, the modulating effects of pretreatment with beta-NF on the induction of sister-chromatid exchanges (SCEs) in V79 cells by mutagens from different chemical classes were investigated in a co-cultivation system consisting of primary chick embryo hepatocytes and V79 Chinese hamster cells. Finally, the effects of pretreatment on benzo[a]pyrene (B(a)P) metabolism were studied in more detail. Pretreatment of cultured primary chick embryo hepatocytes with beta-NF resulted in a large increase in cytochrome P-450 content (a 2.8-fold increase after 31 h). Pretreatment with beta-NF had no effect on the level of SCEs induced by N-nitroso-dimethylamine (NDMA) and 2-aminoanthracene (2AA). Pretreatment with beta-NF resulted in a decrease in B(a)P-induced SCEs. This inhibitory potential was positively related to the beta-NF dose. However, there was an inverse relationship between the inhibitory action of beta-NF and the dose of B(a)P, at higher doses less inhibition was observed. When beta-NF was applied simultaneously with B(a)P the percentage of decrease was about the same as for pretreatment. Pretreatment with beta-NF followed by simultaneous application of beta-NF and B(a)P did not result in larger effects. In addition, subcellular fractions were prepared from chick embryos pretreated with beta-NF in ovo. The use of the S9 fraction resulted in a large decrease (80%) in the induction of SCEs in V79 cells by B(a)P whereas the use of the microsomal fraction resulted in a 70% increase in SCE induction compared with non-pretreated microsomes. Pretreatment with beta-NF in ovo gave rise to a large increase in aryl hydrocarbon hydroxylase (AHH) activity in the hepatic microsomal fraction. Increases were observed in the formation of all B(a)P metabolites. In particular the formation of the proximate carcinogenic and mutagenic metabolite B(a)P-7,8 dihydrodiol was increased 7-fold. The data strongly suggest that the inhibitory effects of pretreatment of cultured primary chick embryo hepatocytes with beta-NF cannot be ascribed to its inducing capabilities but instead seem to be due to the formation of an intracellular pool of beta-NF which acts as a competitive inhibitor for B(a)P metabolism.     

Angiostatic activity and metabolism of cortisol in the chorioallantoic membrane (CAM) of the chick embryo.

There is considerable interest in the discovery of compounds which inhibit angiogenesis dependent (neovascular) diseases. The chick embryo, due to the rapid development of an extensive vascular capillary network in the chorioallantoic membrane (CAM), has been used extensively as a model for studying angiogenesis. Angiostatic steroids are a new class of compounds which inhibit the growth of new capillaries in the chick CAM and in other models of neovascularization. Despite the potential therapeutic importance of these compounds, little is known about the ability of the CAM to metabolize these steroids. We have evaluated the ability of the chick CAM to metabolize cortisol which is both an angiostatic steroid as well as a glucocorticoid. When CAM homogenate was incubated with [3H]cortisol and NADPH at 37 degrees C and pH 7.4, and the reaction products analyzed by reverse phase HPLC, [3H]cortisol was converted exclusively to 20 beta-dihydrocortisol (4-pregnen-11 beta,17 alpha,20 beta,21-tetrol-3-one). The cortisol metabolite, 20 beta-dihydrocortisol, has very little glucocorticoid activity, but shows significant angiostatic activity in the CAM comparable to cortisol. The apparent Km determined for cortisol metabolism was 12 microM and the observed Vmax was 1.4 mumol cortisol/mg protein/min. The majority of the 20 beta-reductase activity was found in the soluble (242,000 g) fraction of CAM homogenate. 20 beta-Reductase activity in chick embryo CAM has not been previously reported.     

17-Epimerization of 17 alpha-methyl anabolic steroids in humans: metabolism and synthesis of 17 alpha-hydroxy-17 beta-methyl steroids.

The 17-epimers of the anabolic steroids bolasterone (I), 4-chlorodehydromethyltestosterone (II), fluoxymesterone (III), furazabol (IV), metandienone (V), mestanolone (VI), methyltestosterone (VII), methandriol (VIII), oxandrolone (IX), oxymesterone (X), oxymetholone (XI), stanozolol (XII), and the human metabolites 7 alpha,17 alpha-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (XIII) (metabolite of I), 6 beta-hydroxymetandienone (XIV) (metabolite of V), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (XV) (metabolite of V), 3'-hydroxystanozolol (XVI) (metabolite of XII), as well as the reference substances 17 beta-hydroxy-17 alpha-methyl-5 beta-androstan-3-one (XVII), 17 beta-hydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (XVIII) (also a metabolite of V), the four isomers 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (XIX) (also a metabolite of VI, VII, and XI), 17 alpha-methyl-5 alpha-androstane-3 beta,17 beta-diol (XX), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (XXI) (also a metabolite of V, VII, and VIII), 17 alpha-methyl-5 beta-androstane-3 beta,17 beta-diol (XXII), and 17 beta-hydroxy-7 alpha,17 alpha-dimethyl-5 beta-androstan-3-one (XXIII) were synthesized via a 17 beta-sulfate that spontaneously hydrolyzed in water to several dehydration products, and to the 17 alpha-hydroxy-17 beta-methyl epimer. The 17 beta-sulfate was prepared by reaction of the 17 beta-hydroxy-17 alpha-methyl steroid with sulfur trioxide pyridine complex. The 17 beta-methyl epimers are eluted in gas chromatography as trimethylsilyl derivatives from a capillary SE-54 or OV-1 column 70-170 methylen units before the corresponding 17 alpha-methyl epimer. The electron impact mass spectra of the underivatized and trimethylsilylated epimers are in most cases identical and only for I, II, and V was a differentiation between the 17-epimers possible. 1H nuclear magnetic resonance (NMR) spectra show for the 17 beta-methyl epimer a chemical shift for the C-18 protons (singlet) of about 0.175 ppm (in deuterochloroform) to a lower field. 13C NMR spectra display differences for the 17-epimeric steroids in shielding effects for carbons 12-18 and 20. Excretion studies with I-XII with identification and quantification of 17-epimeric metabolites indicate that the extent of 17-epimerization depends on the A-ring structure and shows a great variation for the different 17 alpha-methyl anabolic steroids.     

Effect of some antineoplastics on metabolic heme pathway

1. The porphyrinogenic ability of several antineoplastics used in the therapy of the different cancers was evaluated. The action of cyclophosphamide, busulfan and 5-fluorouracil on the amount and nature of the accumulated hepatic porphyrins and on the activity of delta-aminolaevulinate synthase (ALA-S), were estimated at different doses and times of drug treatment in 17-day-old chick embryos. 2. It was observed that cyclophosphamide produces a significant increase in the accumulation of hepatic porphyrins at different doses as well as in the activity of the ALA-S, at all the incubation times. Cyclophosphamide alters the pattern of porphyrins accumulated in the liver, where a coproporphyrin: protoporphyrin ratio higher than in the controls can be observed. 3. Busulfan increased the hepatic porphyrins accumulated in the liver but to a lesser degree than cyclophosphamide. 4. 5-Fluorouracil did not modify the hepatic porphyrin content when it was administered at doses up to 40 mg/embryo. 5. When the embryos were injected with busulfan or 5-fluorouracil no significant differences were observed in the activity of ALA-S up to 11 hr of incubation. 6. These results indicate that cyclophosphamide has a remarkable porphyrinogenic capacity in chick embryo while busulfan, notwithstanding the fact that it alters the haem pathway, it does so to a degree that does not impair the regulation of ALA-S activity. Fluorouracil seems to be non porphyrinogenic in this system, up to 40 mg/embryo.     

Stable isotope studies on steroid metabolism and kinetics: sulfates of 3 alpha-hydroxy-5 alpha-pregnane derivatives in human pregnancy

The metabolism and production rates of 3 alpha-hydroxy-5 alpha-pregnan-20-one sulfate and the 3-sulfate and 3,20-disulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol in pregnant women were studied. The steroid sulfates were labeled with deuterium in the 3 beta,11,11- or 3 beta,11,11,20 beta-positions and were injected intravenously. The deuterium content of steroids in the monosulfate and disulfate fraction of plasma collected at different times after the injection was determined by capillary column gas chromatography/mass spectrometry. The injected steroid sulfates underwent oxidoreduction at C-20 and 16 alpha-hydroxylation. In addition, the 3-sulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol became hydroxylated at C-21. The pregnanediol and pregnanetriol monosulfates were also converted to disulfates. No evidence was obtained for a metabolic sequence involving hydrolysis, oxidoreduction, and resulfation at the C-3 position. Production rates and rates of metabolic transformations were determined using different one- and two-pool models. The production rate of the pregnanolone/pregnanediol monosulfate couple was 0.08 to 0.5 mmol/24 h, the variability probably depending both on individual factors and stage of pregnancy. The half-life time for oxidation and reduction at C-20 was 0.1 to 0.4 hours, reduction being the faster process. The half-life time for the turnover of the steroid skeleton was 1.3 to 3.3 hours. The injected steroid monosulfates were 16 alpha-hydroxylated at a rate of 1 to 8 mumol/24 h. A significant fraction of these 16 alpha-hydroxylated steroid sulfates, 0.5 to 25 mumol/24 h, was formed from other, probably unconjugated, precursors. The 16 alpha-hydroxylated steroid monosulfates underwent rapid oxidoreduction at C-20. The 3-sulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol was hydroxylated at C-21. The production rate of 5 alpha-pregnane-3 alpha,20 alpha,21-triol 3-sulfate was 8 to 36 mumol/24 h in four women and 180 mumol/24 h in one woman, and this steroid was not formed from other precursors to a significant extent. 5 alpha-Pregnane-3 alpha,20 alpha-diol disulfate was a metabolic end product accounting for a major part of the elimination of the steroids injected. Its half-life time was 1.4 to 2.8 hours. The results show that the formation of sulfated steroids with a 3 alpha-hydroxy-5 alpha configuration may account for 50% of the metabolism of progesterone in late pregnancy.     

Differences in steroid specificity for rat androgen binding protein and the cytoplasmic receptor.

Two proteins in the rat, androgen binding protein (ABP) and the cytoplasmic receptor (CR), have high affinity and limited capacity for binding androgens. To determine the structural requirements for binding with high affinity, each protein was partially purified and the ability of over 100 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding sites was assessed. The results indicate marked differences in the steroid specificities of the two proteins. Some alterations of dihydrotestosterone at C-2 or C-2 and C-3 increase binding to ABP two to four-fold. Similarly, the affinity of 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one for ABP increases two-fold when a double bond is created at C-14. Addition of a methyl group in the alpha position at C-7 or C-17, or an ethinyl group at C-17 cause little change in affinity; however, modifications at C-11 and C-17 beta, and deletion of the methyl group at C-10 significantly impair binding to ABP. Binding to the CR is maintained or increased by deletion of the methyl group at C-10. Binding is lessened by modifications at C-3 and C-17 beta. Most alterations at C-2, C-7, C-11, and C-17 alpha have only minor effects on binding to the CR. These studies should provide a molecular basis for predicting the effects of specific structural modifications. When some modifications at C-2 or C-2 and C-3 are combined with changes at C-17 beta, the resulting steroids retain very high affinity for ABP and very limited binding to the CR. Such steroids may provide a means for assessing the function of ABP.     

The 1986 Upjohn award lecture. Interaction of chemicals with hemoproteins: implications for the mechanism of action of porphyrinogenic drugs and nitroglycerin

The ferrochelatase inhibitory activity of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) was studied in chick embryo liver cells. The ferrochelatase inhibitory activity of the 4-butyl, 4-pentyl, and 4-hexyl analogues was considered to be due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of the corresponding N-alkylporphyrins. The relative ferrochelatase inhibitory activity of the DDC analogues has implications for a postulated model of the binding of porphyrins in the ferrochelatase active site. 3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) was shown to be a potent porphyrinogenic agent and to inhibit ferrochelatase in chick embryo liver cells. A related sydnone, 3-benzyl-4-phenylsydnone did not inhibit ferrochelatase activity. These results supported the idea that the porphyrinogenicity of TTMS was due to catalytic activation by cytochrome P-450 leading to heme alkylation and formation of N-vinylprotoporphyrin which inhibits ferrochelatase. Polychlorinated biphenyls, phenobarbital, nifedipine, and a large number of structurally different chemicals which are porphyrinogenic in chick embryo liver cells inhibit uroporphyrinogen decarboxylase by an unknown mechanism. Thus drug-induced porphyrin biosynthesis in chick embryo liver cell culture appears to be caused by inhibition of either ferrochelatase or uroporphyrinogen decarboxylase. The biotransformation of nitroglycerin by human red blood cells is due to a combination of a sulfhydryl-dependent enzymatic process and an interaction with reduced hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone and prolactin are both required for suppression of the induction of rat alpha-lactalbumin activity

Progesterone prevents lactation during pregnancy. This anti-lactogenic effect includes suppression of the advent of alpha-lactalbumin activity, an effect which prevents the formation of lactose. Alpha lactalbumin activity can be induced to some extent in pregnant rat mammary explants by insulin and hydrocortisone alone, and to a greater extent with prolactin in addition, or with EGF in addition. Physiological levels of progesterone markedly inhibit the induction in the presence of prolactin plus insulin and hydrocortisone, only weakly inhibit in the presence of insulin and hydrocortisone alone, and have no inhibitory effect in the presence of EGF plus insulin and hydrocortisone. Prolactin permits some inhibition in the presence of EGF. The results suggest that progesterone does not subvert the essential insulin or glucocorticoid signals. It also appears that transduction of the prolactin signal is required in order that progesterone effectively block induction of alpha-lactalbumin activity.     

The effects of dihydropyridine calcium antagonists on heme biosynthesis in chick embryo liver cell culture

A variety of 1,4-dihydropyridine calcium antagonists were tested for porphyrinogenic activity in chick embryo liver cell culture. 3,5-Dimethoxycarbonyl-1,4-dihydro-2, 6-dimethyl-4-(ortho-nitrophenyl)pyridine (nifedipine) was shown to be a potent porphyrinogenic agent. This activity was shared by a number of related analogues, viz., the 4-phenyl, 4-(meta-nitrophenyl), 4-(para-nitrophenyl), 4-(ortho-methoxyphenyl), 4-(meta-trifluoromethylphenyl), and 4-(para-trifluoromethylphenyl) analogues and nitrendipine; nicardipine exhibited very weak activity. The porphyrinogenic potency of the 1,4-dihydropyridines did not parallel their calcium antagonist activity. Nifedipine did not exhibit ferrochelatase-lowering activity in chick embryo liver cell culture and uroporphyrin and heptacarboxylic acid porphyrin were the major porphyrins to accumulate. Nifedipine did not cause suicidal destruction of cytochrome P-450 in chick embryo hepatic microsomes. Because nifedipine possesses comparable porphyrinogenic activity to sodium secobarbital in chick embryo liver cell culture, caution is required if nifedipine or related drugs are administered to patients with hereditary hepatic porphyria.