Journal
of Sterord
Bmchemisfry,
1978, Vol.
9, pp. 29-32.
Pergamon
TESTOSTERONE
Printed
Press.
in Great
Britam
METABOLISM LUNG TISSUE
BY HUMAN
LEON MILEWICH, PATRICE STEPHENSPARKER and PAUL C. MACDONALD Cecil H. and Ida Green Center for Reproductive Biology Sciences and The Department of Obstetrics and Gynecology, The University of Texas Health Science Center, Southwestern Medical School at Dallas, Dallas, TX 75235, U.S.A. (Received
20 May 1977)
SUMMARY The metabolism of [7-3H(N)l-testosterone
by slices of human lung tissue obtained from an adult male at surgery was studied in a time course experiment. The 17/Ghydroxysteroid oxidoreductase activity of human lung was expressed in the synthesis of isotope-labeled 4-androstene-3,17-dione, 5a-androstane-3,17-dione, androsterone and isoandrosterone. The rate of formation of 4-androstene-3,17-dione followed a linear course for about five min (160pmo1/100mg protein/mm), while the formation of the other 17-oxosteroid metabolites followed a linear course for about 1 h (5a-androstane-3,17-dione, 80 pmol/lOO mg protein/h; androsterone, 2.4 pmol/lOO mg protein/h; isoandrosterone, -28 pmol/lOO mg protein/h). Thus, human lung tissue, in vitro, expresses significant 17/I-hydroxysteroid oxidoreductase activity and, notably, a cofactor milieu that favors the 17-0~0 over the 17ghydroxysteroid oxidoreduction state. This obtains since 5x-dihydrotestosterone formation constituted a very small fraction of total 17-oxosteroid metabolites (- 4.6 pmol/lOO mg protein/h us. 2,832 pmol/lOO mg protein/h). In addition to 17/&hydroxysteroid oxidoreductase activity, the human lung has demonstrable See-reductase, 3/Ghydroxysteroid oxidoreductase and 3a-hydroxysteroid oxidoreductase activities, findings which support the concept that the lung is a site of metabolism of plasma C,,-steroids.
INTRODUCTION
EXPERIMENTAL
The metabolism of tritium-labeled dehydroisoandrosterone and androstenedione by human lung tissue has been reported [l]. The major metabolites of dehydroisoandrosterone were androstenedione and 7a-hydwhile roxydehydroisoandrosterone. testosterone, 5-androstene-3P,17fl-diol, SE-androstanedione* and androsterone were produced in lesser amounts. The products isolated from incubation of androstenedione were testosterone, Sa-androstanedione, androsterone, and isoandrosterone. Since the human lung is engaged in a variety of nonrespiratory functions involving biologically active substances [2] and has a very large blood supply[3] and capillary surface area [4], the lung has the potential to inactivate many substances in the pulmonary circulation, as well as to transform inactive compounds into biologically active substances which could exert their activities in situ or systemically. Since testosterone circulates in the peripheral blood of men and women, we investigated the metabolic role of the human lung in the synthesis of testosterone metabolites with or without hormone activity. The purpose of this study was to evaluate the metabolism of tritium-labeled testosterone by human lung tissue slices, as a function of incubation time.
Normal human lung tissue from the lower lobe was obtained from a 61-year-old white man undergoing left pneumonectomy for keratinizing epidermoid carcinoma which was confined to the left upper lobe. The lung tissue was rinsed in chilled 0.9% sodium chloride solution, blotted, and cut into slices weighing approximately 100mg each. About one hour transpired between surgical removal of the lung tissue and initiation of the incubations. . Solvents and reagents were as previously described
Cll. Steroids. [7-3H(N)]-Testosterone (S.A. 25 Ci/mmol), [4-‘4CJ-testosterone, and [4-14C]-androstenedione (S.A. 55 mCi/mmol), and [4-i4C]-Sa-dihydrotestosterone (S.A. 50mCi/mmol) were purchased from New England Nuclear Corp. and were purified by celiteethylene glycol column chromatography [S] prior to use. [4-i4C]-Sa-androstanedione, [4-‘4C]-androsterone and [4-i4C]-isoandrosterone (S.A. 50 mCi/mmol) were synthesized [6]. Nonradioactive steroids were & purchased from Steraloids, Inc. Incubation procedure. Incubations were carried out in a manner similar to that previously described [l]. In brief, lung tissue slices were incubated with a standard mixture consisting of [7-3H(N)]-testosterone (215 nM, containing 1.22 x lo6 d.p.m.), glucose (3.7 mM) and Krebs-Ringer phosphate buffer, pH 7.4, in a total vol. of l.Oml. Cofactors were not added. Incubation mixtures without tissue or with tissue previously heated in boiling water for 10 min were used as controls. The incubations were carried out at 37”C,
*The following trivial names and abbreviations are used: See-And, 5a_androstanedione, 5a-androstane-3, 17-dione; isoandrosterone, 3p-hydroxy-5z-androstan-17one; Sa-DHT, Sot-dihydrotestosterone, 17fi-hydroxy-5aandrostan-3-one; 4-And, androstenedione, 4-androstene-3.17-dione. 29
30
Ll.Oii MILI M’IC’H
of Sterord
Bmchemisfry,
1978, Vol.
9, pp. 29-32.
Pergamon
TESTOSTERONE
Printed
Press.
in Great
Britam
METABOLISM LUNG TISSUE
BY HUMAN
LEON MILEWICH, PATRICE STEPHENSPARKER and PAUL C. MACDONALD Cecil H. and Ida Green Center for Reproductive Biology Sciences and The Department of Obstetrics and Gynecology, The University of Texas Health Science Center, Southwestern Medical School at Dallas, Dallas, TX 75235, U.S.A. (Received
20 May 1977)
SUMMARY The metabolism of [7-3H(N)l-testosterone
by slices of human lung tissue obtained from an adult male at surgery was studied in a time course experiment. The 17/Ghydroxysteroid oxidoreductase activity of human lung was expressed in the synthesis of isotope-labeled 4-androstene-3,17-dione, 5a-androstane-3,17-dione, androsterone and isoandrosterone. The rate of formation of 4-androstene-3,17-dione followed a linear course for about five min (160pmo1/100mg protein/mm), while the formation of the other 17-oxosteroid metabolites followed a linear course for about 1 h (5a-androstane-3,17-dione, 80 pmol/lOO mg protein/h; androsterone, 2.4 pmol/lOO mg protein/h; isoandrosterone, -28 pmol/lOO mg protein/h). Thus, human lung tissue, in vitro, expresses significant 17/I-hydroxysteroid oxidoreductase activity and, notably, a cofactor milieu that favors the 17-0~0 over the 17ghydroxysteroid oxidoreduction state. This obtains since 5x-dihydrotestosterone formation constituted a very small fraction of total 17-oxosteroid metabolites (- 4.6 pmol/lOO mg protein/h us. 2,832 pmol/lOO mg protein/h). In addition to 17/&hydroxysteroid oxidoreductase activity, the human lung has demonstrable See-reductase, 3/Ghydroxysteroid oxidoreductase and 3a-hydroxysteroid oxidoreductase activities, findings which support the concept that the lung is a site of metabolism of plasma C,,-steroids.
INTRODUCTION
EXPERIMENTAL
The metabolism of tritium-labeled dehydroisoandrosterone and androstenedione by human lung tissue has been reported [l]. The major metabolites of dehydroisoandrosterone were androstenedione and 7a-hydwhile roxydehydroisoandrosterone. testosterone, 5-androstene-3P,17fl-diol, SE-androstanedione* and androsterone were produced in lesser amounts. The products isolated from incubation of androstenedione were testosterone, Sa-androstanedione, androsterone, and isoandrosterone. Since the human lung is engaged in a variety of nonrespiratory functions involving biologically active substances [2] and has a very large blood supply[3] and capillary surface area [4], the lung has the potential to inactivate many substances in the pulmonary circulation, as well as to transform inactive compounds into biologically active substances which could exert their activities in situ or systemically. Since testosterone circulates in the peripheral blood of men and women, we investigated the metabolic role of the human lung in the synthesis of testosterone metabolites with or without hormone activity. The purpose of this study was to evaluate the metabolism of tritium-labeled testosterone by human lung tissue slices, as a function of incubation time.
Normal human lung tissue from the lower lobe was obtained from a 61-year-old white man undergoing left pneumonectomy for keratinizing epidermoid carcinoma which was confined to the left upper lobe. The lung tissue was rinsed in chilled 0.9% sodium chloride solution, blotted, and cut into slices weighing approximately 100mg each. About one hour transpired between surgical removal of the lung tissue and initiation of the incubations. . Solvents and reagents were as previously described
Cll. Steroids. [7-3H(N)]-Testosterone (S.A. 25 Ci/mmol), [4-‘4CJ-testosterone, and [4-14C]-androstenedione (S.A. 55 mCi/mmol), and [4-i4C]-Sa-dihydrotestosterone (S.A. 50mCi/mmol) were purchased from New England Nuclear Corp. and were purified by celiteethylene glycol column chromatography [S] prior to use. [4-i4C]-Sa-androstanedione, [4-‘4C]-androsterone and [4-i4C]-isoandrosterone (S.A. 50 mCi/mmol) were synthesized [6]. Nonradioactive steroids were & purchased from Steraloids, Inc. Incubation procedure. Incubations were carried out in a manner similar to that previously described [l]. In brief, lung tissue slices were incubated with a standard mixture consisting of [7-3H(N)]-testosterone (215 nM, containing 1.22 x lo6 d.p.m.), glucose (3.7 mM) and Krebs-Ringer phosphate buffer, pH 7.4, in a total vol. of l.Oml. Cofactors were not added. Incubation mixtures without tissue or with tissue previously heated in boiling water for 10 min were used as controls. The incubations were carried out at 37”C,
*The following trivial names and abbreviations are used: See-And, 5a_androstanedione, 5a-androstane-3, 17-dione; isoandrosterone, 3p-hydroxy-5z-androstan-17one; Sa-DHT, Sot-dihydrotestosterone, 17fi-hydroxy-5aandrostan-3-one; 4-And, androstenedione, 4-androstene-3.17-dione. 29
30
Ll.Oii MILI M’IC’H
