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.     

Metabolism of androstenedione by human ovarian tissues in vitro with particular reference to reductase and aromatase activity

The ability of granulosa and theca cells of the human ovarian follicle at different stages of development, as well as stromal and luteal tissues from human ovaries to metabolize androstenedione (delta 4) to testosterone (T), dihydrotestosterone (DHT), estrone (E1) and estradiol (E2) with or without exposure to additional amounts of folicle-stimulating hormone was investigated by in vitro experiments. The results show that all the aforementioned ovarian tissues metabolized delta 4 to DHT. Indeed, with the exception of estrogen-secreting granulosa cells from large antral follicle (greater than 10 mm diameter) and possibly also luteal tissue from mid-luteal phase ovaries, the various ovarian tissues preferentially metabolized delta 4 to DHT instead of E (E1 + E2). Although thecal tissue is a major source of delta 4 in human ovaries it is concluded that the granulosa cells do not interact with the theca for the synthesis of E as the follicle enlarges from 1 to 10 mm in diameter. Indeed, excessive thecal delta 4 during this growth phase probably inhibits normal follicular development. However, as the follicle enlarges beyond 10 mm in diameter, and as the granulosa cells begin to preferentially metabolize delta 4 to E, the two cell-types of the follicle may increasingly interact to enhance the follicular output of E.     

Metabolism of testosterone in human lung in vitro

• 1.1. The metabolism of 4-¹⁴C-testosterone in human lung in vitro was investigated.
• 2.2. The metabolism was most pronounced in incubations of homogenated tissue, whereas it was rather restricted in the mitochondrial, microsomal and soluble fraction incubations.
• 3.3. The by far most prominent metabolite in all experiments was androst-4-ene-3,17-dione.
• 4.4. No slfate or glucuronide conjugation took place.

     

Metabolism of the contraceptive steroid desogestrel by human liver in vitro

The metabolism of the progestogen oral contraceptive desogestrel (Dg) has been studied in vitro using human liver microsomes. Metabolites have been separated using radiometric high performance liquid chromatography and identified by co-chromatography with authentic standards and by mass spectrometry. All the livers examined (n = 6) were able to form 3-keto desogestrel as the main identifiable metabolite and also the presumed intermediates 3 alpha-hydroxydesogestrel (3 alpha-OHDg) and 3 beta-hydroxydesogestrel (3 beta-OHDg). In addition, a large polar heterogenous peak was evident on the radiochromatograms which did not co-chromatograph with any known metabolites of desogestrel. Inter-individual variability in metabolite formation was seen. A number of drugs were examined for their propensity to inhibit desogestrel metabolism. Primaquine was the most potent tested having an IC50 value (inhibitory concentration reducing overall metabolite production by 50%) of 30 microM. Cimetidine, trilostane and levonorgestrel failed to inhibit at 250 microM. With 3 alpha-OHDg as substrate, 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH) activity was 1.0 +/- 0.3 nmol min-1 mg-1 protein which was five times greater than the activity of the 3 beta-HSDH towards 3 beta-OHDg. Miconazole was the most potent inhibitor tested having IC50 values of 14 and 95 microM for 3 alpha- and 3 beta-HSDH respectively. Surprisingly, trilostane was without inhibitory effect on either enzyme, which contrasts with other data involving 3 beta-HSDH in steroidogenic tissue. Our observations with trilostane may reflect tissue differences in the enzyme and/or differences in endogenous vs exogenous steroids (i.e. in the conversion of 3 beta-OHDg to 3-ketodesogestrel there is no requirement for isomerization). Kinetic parameters of 3 alpha-HSDH were also determined.     

Excretion of non-conjugated androstenedione and testosterone in human urine.

A method for the measurement of unconjugated testosterone and androstenedione in human urine is described. The method uses chromatographic separation followed by radioimmunoassay and has been examined for reliability. The mean 24-hour excretion of androstenedione by adult male subjects was 2.5 mug and of testosterone was 0.8 mug. For women, the mean excretion was 2.9 mug of androstenedione and 0.25 mug of testosterone. In pregnancy, androstenedione excretion was occasionally elevated above the normal range, but testosterone excretion was quite commonly increased. Some hirsute subjects exhibited an increase in androstenedione excretion, which was decreased by administration of dexamethasone. The results suggest that the amount of unconjugated testosterone in urine is not a direct reflection of the plasma free testosterone, but urinary androstenedione may be a useful reflection of plasma androstenedione levels.     

Metabolism of testosterone in human,rat and rabbit fetal lungs and in rabbit neonatal lung in vitro

• 1.1. Metabolism of 4-¹⁴C-testosterone was investigated in human, rat and rabbit fetal lung subcellular fractions and also in rabbit neonatal lungs. Androst-4-ene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and both 5α- and 5β-androstane-3α,17β-diols were identified as metabolites of testosterone.
• 2.2. The microsomal fraction produced mainly 5α-reduced epimers while the cytosol incubations resulted in 5β-reduced metabolites.
• 3.3. No conjugation was found.
• 4.4. The amounts of polar metabolites in the microsomal incubations and the amounts of dihydroxy-lated metabolites in the soluble fraction incubations were statistically significantly greater in the neonatal rabbit lung incubations compared with those of fetal lungs.

     

Metabolism of 11-oxygenated steroids. Metabolism in vitro by preparations of liver

1. The isolation and partial purification of 11beta-hydroxy steroid dehydrogenase from rat and guinea-pig liver microsomes has been achieved by conventional methods. 2. The efficiency of different 11-oxygenated steroids as substrates has been examined. The relative efficiencies confirm in the main the stereochemical theory of the enzyme-coenzyme-substrate complex that was proposed earlier on the basis of studies in vivo. Delta(4)-3-Ketones and 5alpha-hydrogen steroids are readily metabolized by the enzyme. 5beta-Hydrogen steroids and Delta(4)-3-ketones with certain large alpha-substituents are metabolized to a limited extent or not at all. Halogen substitution in the 9alpha-position enhances the rate of reduction of 11-ketones but blocks the oxidation of the related 11beta-ols. 3. 9alpha-Fluorocortisol is a competitive inhibitor of the oxidation of cortisol, but 9alpha-fluorocortisone is reduced at five to ten times the initial velocity of cortisone. 4. 11beta-Hydroxy steroid dehydrogenase activity has been found in liver microsomes of rat, guinea pig, rabbit and calf. 5. Relative substrate efficiencies and K(m) values are similar in whole (debris-free) homogenates, washed microsomes and acetone-dried powders of washed microsomes. 6. A variety of conditions have been examined for the observation of 11beta-hydroxy steroid dehydrogenase activity. NADP(H) is an efficient and NAD(H) a very poor coenzyme for the reaction.     

Metabolism of environmental pollutants by the isolated perfused lung.

An isolated perfused rabbit lung system was developed for the study of pulmonary metabolism of foreign compounds. The main features of the system include the use of autologous whole blood, constant pressure perfusion, subatmospheric ventilation, and measurement of a variety of physiological and biochemical parameters. Pulmonary metabolism of benzo(a)pyrene has been investigated with this system. In addition to the 3-hydroxy metabolite, three dihydrodiols and an unidentified polar metabolite were also found. The polar metabolite accounted for approximately 50% of all metabolites found in the five compartments of the perfusion system. Pretreatment with 3-methylcholanthrene increased total metabolism of benzo(a)pyrene and shifted the pattern of metabolites. The perfusion system for the rabbit has been extensively modified for use with rats and guinea pigs. These smaller animals are currently being used to investigate pulmonary metabolism of trichloroethylene.     

The aromatization of androstenedione by human adipose and liver tissue

The ability of adipose tissue, obtained from women at operation and adipose tissue and liver obtained 4 h after the death of a 4 y old girl, to metabolise androstenedione was investigated. There was significant conversion of androstenedione to oestrone (0.18%) by liver tissue. Conversion of androstenedione to oestrone was also detected (range 0.02–0.60%) in five of nine samples of adipose tissue obtained from the abdominal wall. The highest conversion of androstenedione to oestrone were seen in one premenopausal woman (0.60%) who had recently ceased treatment for hyperprolactinaemia and a postmenopausal woman (0.55%) who had endometrial cancer. Three samples of adipose tissue obtained from the intraperitoneal cavity all had the ability to convert androstenedione to oestrone, although conversion by adipose tissue obtained from the intraperitoneal cavity of the subject with endometrial cancer (0.02%) was much lower than that of adipose tissue obtained from the abdominal wall (0.55%). Non-phenolic products were also isolated after incubation of androstenedione with adipose tissue and preliminary investigation by paper chromatography showed that two of the products had R_F values identical with testosterone and dihydrotestosterone respectively.     

Direct analysis of dehydroisoandrosterone in plasma.

DHA (1) has been measured in plasma by a radioimmunoassay procedure using an antiserum to DHA-7-BSA whose specificity is such that the procedure is carried out directly on diluted, unextracted plasma. The method has been used to obtain plasma DHA secretory patterns and mean concentrations and the data are in accord with those determined by related but more laborious techniques.