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Cutaneous Androgen Metabolism: Basic Research and Clinical Perspectives

      The skin, especially the pilosebaceous unit composed of sebaceous glands and hair follicles, can synthesize androgens de novo from cholesterol or by locally converting circulating weaker androgens to more potent ones. As in other classical steroidogenic organs, the same six major enzyme systems are involved in cutaneous androgen metabolism, namely steroid sulfatase, 3β-hydroxy-steroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, steroid 5α-reductase, 3α-hydroxysteroid dehydrogenase, and aromatase. Steroid sulfatase, together with P450 side chain cleavage enzyme and P450 17-hydroxylase, was found to reside in the cytoplasm of sebocytes and keratinocytes. Strong steroid sulfatase immunoreacti-vity was observed in the lesional skin but not in unaffected skin of acne patients. 3β-hydroxysteroid dehydrogenase has been mainly immunolocalized to sebaceous glands, with the type 1 being the key cutaneous isoenzyme. The type 2 17β-hydroxysteroid dehydrogenase isoenzyme predominates in sebaceous glands and exhibits greater reductive activity in glands from facial areas compared with acne nonprone areas. In hair follicles, 17β-hydroxysteroid dehydrogenase was identified mainly in outer root sheath cells. The type 1 5α-reductase mainly occurs in the sebaceous glands, whereby the type II isoenzyme seems to be localized in the hair follicles. 3α-hydroxysteroid dehydrogenase converts dihydrotestosterone to 3α-androstanediol, and the use of 3α-androstanediol glucuronide serum level to reflect the hyperandrogenic state in hirsute women may be a reliable parameter, especially for idiopathic hirsutism. In acne patients it is still controversial if 3α-androstanediol glucuronide or androsterone glucuronide could serve as suitable serum markers for measuring androgenicity. Aromatase, localized to sebaceous glands and to both outer as well as inner root sheath cells of anagen terminal hair follicles, may play a “detoxifying” role by removing excess androgens. Pharmacologic development of more potent specific isoenzyme antagonists may lead to better clinical treatment or even prevention of androgen-dependent dermatoses.

      Keyword

      3α-Adiol
      5α-androstane-3α, 17β-diol (3α-androstanediol)
      DHEA
      dehydroepiandrosterone
      DHEA-S
      dehydroepiandrosterone-sulfate
      5α-DHT
      5α-dihydrotestosterone
      DP
      dermal papilla
      3β-Δ5-HSD
      3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase
      NAD
      nicotinamide adenosine dinucleotide
      NADP
      nicotinamide adenosine dinucleotide pyrophosphatase
      ORS
      outer root sheath cells
      Sex hormones, like other steroid hormones, are synthesized from their major parental precursor cholesterol, which undergoes side chain cleavage by the mitochon-drial P450 side chain cleavage enzyme (P450scc) to form Δ5-pregnenolone, releasing a C6 aldehyde. Δ5-pregnenolone, the required intermediate compound in the synthesis of all steroid hormones, can be converted intracellularly to progesterone by the action of 3β-ol-dehydrogenase/Δ4,5-isomerase or to 17α-hydroxypregnenolone by 17α-hydroxylase. The ring system “sterane” of the steroid hormones, carrying a cyclopentanoperhydrophenanthrene nucleus, is a stable structure that cannot be broken down by mammalian cells. Conversion of active hormones to less active or inactive forms involves alteration of ring substituents rather than the ring structure itself. Sex hormones can be easily distinguished by the carbon numbers, C-19 being androgens, C-18 being estrogens, and C-21 being progestenoids. Androgens are derivatives of androstane and contain either a keto group [e.g., dehydroepiandrosterone (DHEA) and androstenedione] or hydroxy group [testosterone and 5α-dihydrotestosterone (5α-DHT)] at position 17 of the ring system.
      Cholesterol synthesis in mammalian systems is regulated by the key enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and it has been confirmed that this process also occurs in the epidermis and the sebaceous glands (
      • Menon G.K.
      • Feingold K.R.
      • Mosr A.H.
      • Brown B.E.
      • Elias P.M.
      De novo sterologenesis in the skin. II. Regulation by cutaneous barrier requirements.
      ;
      • Smythe C.D.
      • Greenall M.
      • Kealey T.
      The activity of HMG-CoA reductase and acetyl-CoA carboxylase in human apocrine sweat glands, sebaceous glands, and hair follicles is regulated by phosphorylation and by exogenous cholesterol.
      ). Recent work demonstrated the cutaneous expression of steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage (P450scc) and cytochrome P450 17α-hydroxylase (P450c17), suggesting the cutaneously derived cholesterol could be further used as a substrate for de novo steroid hormone synthesis in human epidermis and the sebaceous gland.
      Billich A, Rot A, Lam C, Schmidt JB, Schuster I: Immunohistochemical localization of steroid sulfatase in acne lesions: Implications for the contribution of dehydroepiandrosterone sulfate to the pathogenesis of acne. Hormone Res 53:99, 2000 (Abstr.)
      ,
      Thiboutot DM, Sivarajah A, Gilliland K, Cong Z, Clawson G: The skin as steroidogenic tissue: Enzymes and cofactors involved in the initial steps of steroidogenesis are expressed in human skin and rat preputial sebocytes. J Invest Dermatol 117:410, 2001 (Abstr.)
      1Billich A, Rot A, Lam C, Schmidt JB, Schuster I: Immunohistochemical localization of steroid sulfatase in acne lesions: Implications for the contribution of dehydroepiandrosterone sulfate to the pathogenesis of acne. Hormone Res 53:99, 2000 (Abstr.)
      2Thiboutot DM, Sivarajah A, Gilliland K, Cong Z, Clawson G: The skin as steroidogenic tissue: Enzymes and cofactors involved in the initial steps of steroidogenesis are expressed in human skin and rat preputial sebocytes. J Invest Dermatol 117:410, 2001 (Abstr.)
      On the other hand, steroid hormones inhibit the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in SZ95 sebocytes at the mRNA level in a negative feedback regulation process (
      • Zouboulis ChC
      • Seltmann H.
      • Neitzel H.
      • Orfanos C.E.
      Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).
      ;2002).
      In classical “central” steroidogenic organs such as the gonads and the adrenal gland, testosterone can be derived mainly from two pathways: (i) progesterone, 17α-hydroxyprogesterone, Δ4-androstenedione, or (ii) 17α-hydroxypregnenolone, DHEA, Δ5-androstenediol (
      • Stauss J.S.
      • Pochi P.E.
      Recent advances in androgen metabolism and their relation to the skin.
      ;
      • Witt B.R.
      • Thorneycroft J.H.
      Reproductive steroid hormones: generation, degradation, reception, and action.
      ). Alternatively, in many “peripheral” organs, such as the skin, the potent tissue androgen testosterone results from the conversion of circulating dehydroepiandrosterone sulfate (DHEA-S), a weak but most abundant androgen in higher primates, through the serial action of steroid sulfatase, 3β-hydro-xysteroid dehydrogenase/Δ5-4 isomerase (3β-Δ5-HSD), and 17β-HSD (
      • Labrie F.
      Intracrinology.
      ;
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ). Testosterone can be further “activated” to the physiologically most potent tissue androgen 5α-DHT through the action of 5α-reductase or “inactivated” to estradiol by aromatase (
      • Kaufman K.D.
      Androgen metabolism as it affects hair growth in androgenetic alopecia.
      ). In contrast to classical endocrinology depicting steroid hormone formation and secretion from classical steroidogenic organs, such as gonads and adrenal glands, this peripheral “on the spot” intracellular hormone synthesis/action, now coined as “intracrinology”, has been shown to take place in various peripheral, hormone-target tissues such as placenta, prostate, adipose tissue, skin, and skin appendages (Figure 1) (
      • Labrie F.
      • Luu-The V.
      • Lin S.X.
      • et al.
      Intracrinology: role of the family of 17 beta-hydroxysteroid dehydrogenases in human physiology and disease.
      ;
      • Zouboulis ChC
      Human skin: An independent peripheral endocrine organ.
      ). The androgen-sensitive skin appendages (sebaceous gland, hair follicle, and sweat gland) each metabolize androgens in a characteristic pattern; however, sweat glands and sebaceous glands account for the vast majority of androgen metabolism in skin (
      • Deplewski D.
      • Rosenfield R.L.
      Role of hormones in pilosebaceous unit development.
      ). In postmenopausal women, 100% of the active sex steroids are synthesized in peripheral target tissues from inactive steroid precursors, whereas, in adult men, approximately 50% of androgens are locally made in intracrine target tissues (
      • Labrie F.
      • Luu-The V.
      • Labrie C.
      • Pelletier G.
      • El-Alfy M.
      Intracrinology and the skin.
      ). Not much is known, however, about the extent to which (i) the de novo cutaneous androgenesis from epidermally formed cholesterol, or (ii) the locally active conversion of potent androgens from circulating DHEA contribute to the androgens formed in the skin.
      Figure thumbnail gr1
      Figure 1Pathways of cutaneous androgen metabolism and the converting enzymes.
      Here is a review of the recent advances in the understanding of cutaneous androgen synthesis and metabolism, trying to correlate the expression of different converting isoenzymes to the pathogenesis of androgen-dependent dermatoses, such as acne vulgaris, hirsutism, and androgenetic alopecia. The current therapeutics and future developments based on this continuously expanding knowledge are also discussed.

      HISTORICAL REVIEW

      The first recognition of the role of androgens in the pathogenesis of cutaneous disorders probably came from Aristotle as early as the fourth century BC, as he noticed the relation between the occurrence of baldness (androgenetic alopecia) and the gender state or the sexual maturity (
      • Montagna W.
      Phylogenetic significance of the skin of man.
      ). It was not until 1942 as Hamilton's pioneering work on castrates subjected to testosterone injections (
      • Hamilton J.B.
      Male hormone is prerequisite and an incitant in common baldness.
      ), which for the first time provided the scientific evidence and hence provoked further investigation on the androgen metabolism in the skin (
      • Takashima I.
      Androgenetic alopecia: pathophysiological aspects in man and animals.
      ).
      The first androgen hormone to be characterized was androsterone isolated from the urine of adult men in 1931 (
      • Tausk M.
      Practically applicable results of twenty years of research in endocrinology.
      ). Testosterone was then demonstrated to be the androgen secreted from testis in 1935. Experimental work on the androgen metabolism in vitro began intensively in 1950s by applying large-scale perfusion (
      • Caspi E.Y.
      • Levy H.
      • Hechter O.M.
      Cortisone metabolism in liver. II. Isolation of certain cortisone metabolites.
      ;
      • Caspi E.Y.
      • Hechter O.M.
      Corticosteroid metabolism in liver. III. Isolation of additional cortisone metabolites.
      ) or by incubating animal internal organ homogenates, e.g., rat liver (
      • Schneider J.J.
      • Horstmann P.M.
      Effects of incubating desoxycorticosterone with various rat tissues.
      ;
      • Taylor W.
      The metabolism of progesterone by animal tissues in vitro. I. Factors influencing the metabolism of progesterone by rat liver and the investigation of the products of metabolism.
      ;
      • Kochakian C.D.
      • Stidworthy G.
      Metabolism of Δ4-androstene-3, 17-dione by tissue homogenates.
      ) with various steroids, such as cortisone, desoxycorticosterone, progesterone, and androstenedione. Metabolites were separated and isolated mainly by thin-layer adsorption chromatography with paper, aluminum or silica gels as stationary phase. During the 1950s and 1960s increasing evidence accumulated that the skin might be one of the sites of peripheral androgen metabolism (
      • Wotiz H.H.
      • Mescon H.
      • Doppel H.
      • Lemon H.M.
      The in vitro metabolism of testosterone by human skin.
      ;
      • Stauss J.S.
      • Pochi P.E.
      Recent advances in androgen metabolism and their relation to the skin.
      ). The presence of different hydroxysteroid dehydrogenases, including 3α-HSD, 3β-HSD, and 17β-HSD, was soon histochemically revealed in the human skin (
      • Baillie A.H.
      • Calman K.C.
      • Milne J.A.
      Histochemical distribution of hydroxysteroid dehydrogenases in human skin.
      ), especially in the sebaceous gland (
      • Baillie A.H.
      • Thomson J.
      • Milne J.A.
      The distribution of hydroxysteroid dehydrogenase in human sebaceous glands.
      ). Active androgen metabolism was further confirmed by incubating human skin with various (radiolabeled) steroids (
      • Wotiz H.H.
      • Mescon H.
      • Doppel H.
      • Lemon H.M.
      The in vitro metabolism of testosterone by human skin.
      ;
      • Gallegos A.J.
      • Berliner D.L.
      Transformation and conjugation of dehydroepiandrosterone by human skin.
      ;
      • Gomez E.C.
      • Hsia S.L.
      In vitro metabolism of testosterone-4-14C and Δ4-androstene-3,17-dione-4-14C in human skin.
      ); i.e., the activity of 17β-HSD in the skin was demonstrated by showing the cutaneous conversion of androstenedione to testosterone or 5α-androstanedione to 5α-DHT (
      • Gomez E.C.
      • Hsia S.L.
      In vitro metabolism of testosterone-4-14C and Δ4-androstene-3,17-dione-4-14C in human skin.
      ). Studies on rat and human prostate showed that 5α-DHT, rather than testosterone, might be the active androgenic hormone at the target site (
      • Farnsworth W.E.
      • Brown J.R.
      Metabolism of testosterone by the human prostate.
      ;
      • Bruchovsky N.
      • Wilson J.D.
      The conversion of testosterone to 5α-androstan-17β-ol-3-one by rat prostate in vivo and in vitro.
      ) and this conversion could also take place in the skin (
      • Wilson J.D.
      • Walker J.D.
      The conversion of testosterone to 5α-androstan-17β-ol-3-one (dihydrotestosterone) by skin slices of man.
      ;
      • Flamigni C.
      • Collins W.P.
      • Koullapis E.N.
      • Craft I.
      • Sommerville I.F.
      Androgen metabolism in human skin.
      ;
      • Bingham K.D.
      • Shaw D.A.
      The metabolism of testosterone by human male scalp skin.
      ). Attempts to inhibit 5α-reductase in the skin cells were soon made (
      • Zerhouni N.A.
      • Maes M.
      • Sultan C.
      • Rothwell S.
      • Migeon C.J.
      Selective inhibition by secosteroids of 5 alpha-reductase activity in human sex skin fibroblasts.
      ;
      • Leshin M.
      • Wilson J.D.
      Inhibition of steroid 5 alpha-reductase from human skin fibroblasts by 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one.
      ). Biochemical characterization of the different androgen converting enzymes, including the enzyme activity, coenzymes, kinetic properties, and intracellular distribution was performed between 1970s and 1980s. Most of these data were based on the studies on congenital adrenal hyperplasia, various virilizing and feminizing syndromes, and hirsutism (
      • New M.I.
      • Dupont B.
      • Pang S.
      • Pollack M.
      • Levine L.S.
      An update of congenital adrenal hyperplasia.
      ;
      • Mauvais-Jarvis P.
      • Kuttenn F.
      • Mowszowicz I.
      Hirsutism.
      ;
      • James V.H.
      • Few J.D.
      Adrenocorticosteroids: chemistry, synthesis and disturbances in disease.
      ). Because of the variable stability of different enzymatic activities in broken cell preparations and the difficulties in purification and stabilization of these membrane-bound enzymes, it was not until the 1990s when the introduction of newer molecular biologic methods, such as RNA expression, cloning, and analysis that better elucidation and comparison of the genetic structure, function, ontogeny, and tissue-specific expression of these converting isoenzymes became feasible (
      • Labrie F.
      Intracrinology.
      ;
      • Russell D.W.
      • Wilson J.D.
      Steroid 5 alpha-reductase: two genes/two enzymes.
      ).

      STEROID SULFATASE

      The steroid sulfatase is a microsomal enzyme widely distributed in human tissues that catalyzes the hydrolysis of sulfated 3-hydroxy steroids to the corresponding free active 3-hydroxy steroids. Early studies in vitro and in vivo have shown that the human steroid sulfatase activity appears after birth, such as in foreskin and infantile abdominal skin (
      • Kim M.H.
      • Herrmann W.L.
      In vitro metabolism of dehydroepiandrosterone sulfate in foreskin, abdominal skin and vaginal mucosa.
      ). Major breakthroughs in the evaluation of cutaneous expression of steroid sulfatase came from the study on its role in X-linked recessive ichthyosis, which has been shown to be associated with the deficiency of the steroid sulfatase/arylsulfatase C (
      • Schlammadinger J.
      • Meyer J.C.
      • Vajda I.
      • Szabo G.
      X-linked recessive ichthyosis. Reinvestigation of a family first described in 1928.
      ;
      • Herrmann F.H.
      [The genetics and molecular genetics of X-chromosomal recessive ichthyosis]. [German].
      ;
      • Herrmann F.H.
      • Wirth B.
      • Wulff K.
      • et al.
      Gene diagnosis in X-linked ichthyosis.
      ). The steroid sulfatase gene is localized to the distal short arm of the X chromosome and most X-linked ichthyosis patients present large deletions of the steroid sulfatase gene and flanking markers, whereas a minority show a point mutation or partial deletion of the steroid sulfatase gene (
      • Bonifas J.M.
      • Epstein Jr, E.H.
      Detection of carriers for X-linked ichthyosis by Southern blot analysis and identification of one family with a de novo mutation.
      ;
      • Basler E.
      • Grompe M.
      • Parenti G.
      • Yates J.
      • Ballabio A.
      Identification of point mutations in the steroid sulfatase gene of three patients with X-linked ichthyosis.
      ;
      • Alperin E.S.
      • Shapiro L.J.
      Characterization of point mutations in patients with X-linked ichthyosis. Effects on the structure and function of the steroid sulfatase protein.
      ;
      • Morita E.
      • Katoh O.
      • Shinoda S.
      • Hiragun T.
      • Tanaka T.
      • Kameyoshi Y.
      • Yamamoto S.
      A novel point mutation in the steroid sulfatase gene in X-linked ichthyosis.
      ;
      • Sugawara T.
      • Shimizu H.
      • Hoshi N.
      • Fujimoto Y.
      • Nakajima A.
      • Fujimoto S.
      PCR diagnosis of X-linked ichthyosis: identification of a novel mutation (E560P) of the steroid sulfatase gene.
      ;
      • Valdes-Flores M.
      • Kofman-Alfaro S.H.
      • Vaca A.L.
      • Cuevas-Covarrubias S.A.
      Deletion of exons 1-5 of the STS gene causing X-linked ichthyosis.
      ). Activity of steroid sulfatase was also demonstrated in cultured keratinocytes (
      • Milewich L.
      • Shaw C.B.
      • Sontheimer R.D.
      Steroid metabolism by epidermal keratinocytes.
      ) in which cholesterol sulfate could inhibit sterol esterification. These data suggest a novel role for cholesterol sulfate as a modulator of cellular lipid biosynthesis (
      • Williams M.L.
      • Rutherford S.L.
      • Feingold K.R.
      Effects of cholesterol sulfate on lipid metabolism in cultured human keratinocytes and fibroblasts.
      ). In patients with X-linked recessive ichthyosis, cholesterol sulfate accumulation rather than cholesterol deficiency seemed to be responsible for the barrier abnormality (
      • Zettersten E.
      • Man M.Q.
      • Sato J.
      • et al.
      Recessive X-linked ichthyosis: role of cholesterol-sulfate accumulation in the barrier abnormality.
      ). On the other hand, in the occipital scalp of normal healthy subjects as well as in the beard, the specific activity of steroid sulfatase was significantly higher in the hair dermal papillae (DP) as compared with that in the connective tissue sheaths or root sheaths (
      • Hoffmann R.
      • Rot A.
      • Niiyama S.
      • Billich A.
      Steroid sulfatase in the human hair follicle concentrations in the dermal papilla.
      ).

      3β-Δ5-HSD

      This enzyme catalyzes an obligatory step in the biosynthesis of androgens, estrogens, mineralocorticoids, and glucocorticoids; the oxidation/isomerization of 3-β-hydroxy-5-ene steroids (Δ5-3β-hydroxysteroids) into the corresponding 3-keto-4-ene steroids (Δ4-3-ketosteroids), i.e., the transformation of DHEA into androstenedione and androstenediol into testosterone, respectively. This process could be seen as the first step to form more potent androgens and to amplify the androgenic effect. 3β-Δ5-HSD is found not only in classic steroidogenic tissues (placenta, adrenal cortex, ovary, and testis), but also in several peripheral tissues, including skin, adipose tissue, breast, lung, endometrium, prostate, liver, kidney, epididymis, and brain (
      • Labrie F.
      • Simard J.
      • Luu-The V.
      • et al.
      Structure and tissue-specific expression of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase genes in human and rat classical and peripheral steroidogenic tissues.
      ). This tissue-specific manner of expression involves separate mechanisms of regulation. An important feature in liver and kidney (at least of hamster, mouse, rabbit, and rat) is the sexual dimorphic nature of 3β-Δ5-HSD. Two types of human 3β-Δ5-HSD cDNA clones have been characterized; the corresponding genes are located in chromosome 1p13.1, containing four exons and three introns with a total length of 7.7–7.8 kbp, and encoding proteins of 371 and 372 amino acids, respectively, which share 93.5% homology and both prefer NAD+ as cofactors (
      • Pelletier G.
      • Dumont E.
      • Simard J.
      • Luu-The V.
      • Belanger A.
      • Labrie F.
      Ontogeny and subcellular localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the human and rat adrenal, ovary and testis.
      ;
      • Labrie F.
      • Belanger A.
      • Simard J.
      • Luu-The V.
      • Labrie C.
      DHEA and peripheral androgen and estrogen formation: Intracrinology.
      ;
      • Simard J.
      • Durocher F.
      • Mebarki F.
      • et al.
      Molecular biology and genetics of 3 beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family.
      ). Human type 1 3β-HSD is the almost exclusive mRNA species expressed in the skin, mammary gland, and placenta (syncytial trophoblast), whereas the type 2 isoform is almost exclusively expressed in the adrenal cortex and the gonads.
      Early studies demonstrated 3β-Δ5-HSD enzyme activity in fibroblasts (
      • Gallegos A.J.
      • Berliner D.L.
      Transformation and conjugation of dehydroepiandrosterone by human skin.
      ), and that the sebaceous gland possessed the highest activity in the human skin (
      • Itami S.
      • Takayasu S.
      Activity of 3 beta-hydroxysteroid dehydrogenase delta 4-5 isomerase in the human skin.
      ;
      • Simpton N.B.
      • Cunliffe W.J.
      • Hodgins M.B.
      The relationship between the vitro activity of 3 beta-hydroxysteroid dehydrogenase delta 4-5 isomerase in human sebaceous glands and their secretory activity in vivo.
      ), which was further confirmed by the immunohistochemical localization of 3β-Δ5-HSD specifically in sebaceous glands (
      • Dumont M.
      • Luu-The V.
      • Dupont E.
      • Pelletier G.
      • Labrie F.
      Characterization, expression, and immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in human skin.
      ;
      • Sawaya M.E.
      • Penneys N.S.
      Immunohistochemical distribution of aromatase and 3 β-hydroxysteroid dehydrogenase in human hair follicle and sebaceous gland.
      ). In cultured skin cells, 3β-Δ5-HSD mRNA was only detected in normal and immortalized sebocytes (SZ95), but neither in normal keratinocytes and in HaCaT cells nor in melanoma cells (MeWo melanoma cells) (
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ). The type 1 isotype of 3β-Δ5-HSD could exclusively be detected. By converting androstenediol to testosterone, an intense enzyme activity of 3β-Δ5-HSD was also revealed in the DP of human terminal hair follicle (
      • Hoffmann R.
      • Rot A.
      • Niiyama S.
      • Billich A.
      Steroid sulfatase in the human hair follicle concentrations in the dermal papilla.
      ). The enzyme activity did not correlate with sebum excretion rate (
      • Simpton N.B.
      • Cunliffe W.J.
      • Hodgins M.B.
      The relationship between the vitro activity of 3 beta-hydroxysteroid dehydrogenase delta 4-5 isomerase in human sebaceous glands and their secretory activity in vivo.
      ) and the enzyme expression did not vary with body site or sex (
      • Sawaya M.E.
      • Penneys N.S.
      Immunohistochemical distribution of aromatase and 3 β-hydroxysteroid dehydrogenase in human hair follicle and sebaceous gland.
      ).

      17B-HSD

      The last and key step in the formation of androgens and estrogens is catalyzed by members of the family of 17β-HSD, whereas the reduction step by 17β-HSD is essential for the formation of the more active androgens and the oxidative reaction inactivates the potent sex steroids. These events take place in the same cell where synthesis occurs. The 17β-HSD thus provide each cell with the means of precisely controlling the intracellular concentration of each sex steroid according to local needs (
      • Zouboulis ChC
      Human skin: An independent peripheral endocrine organ.
      ). To date, seven types of human 17β-HSD have been cloned, sequenced, and characterized, designated types 1–7 in the chronologic order of their isolation (
      • Biswas M.G.
      Russell DW: Expression cloning and characterization of oxidative 17beta- and 3alpha-hydroxysteroid dehydrogenases from rat and human prostate.
      ;
      • Labrie F.
      • Luu-The V.
      • Lin S.X.
      • Labrie C.
      • Simard J.
      • Breton R.
      • Bélanger A.
      The key role of 17β-hydroxysteroid dehydrogenases in sex steroid biology.
      ;
      • Krazeisen A.
      • Breitling R.
      • Imai K.
      • Fritz S.
      • Moller G.
      • Adamski J.
      Determination of cDNA, gene structure and chromosomal localization of the novel human 17beta-hydroxysteroid dehydrogenase type 7(1).
      ;
      • Pelletier G.
      • Luu-The V.
      • Tetu B.
      • Labrie F.
      Immunocytochemical localization of type 5 17 beta-hydroxysteroid dehydrogenase in human reproductive tissues.
      ). The type 1 17β-HSD (17β-HSD1), encoded in chromosome 17q21, is a cytosolic enzyme that was found in ovary, placenta, and in breast cancer cells. 17β-HSD2, encoded in chromosome 16q24, is a microsomal enzyme that was found in placenta, endometrium, normal breast cells, prostate, liver, small intestine, kidney, pancreas, and colon. 17β-HSD3, encoded in chromosome 9q22, was only detected in testis and its importance in male steroid hormone physiology was underscored by its deficiency in a form of male pseudohermaphroditism, the only genetic mutation of these group described until now (
      • Andersson S.
      • Moghrabi N.
      Physiology and molecular genetics of 17β-hydroxysteroid dehydrogenase.
      ). 17β-HSD4 is a peroxisomal enzyme with its mRNA mainly expressed in liver, heart, prostate, testis, and prostate cancer cell lines. 17β-HSD5, encoded in chromosome 10p14,15, was found in the placenta, ovary, endometrium, mammary gland, testis, liver, skeletal muscle, and skin (
      • Labrie F.
      • Luu-The V.
      • Lin S.X.
      • Labrie C.
      • Simard J.
      • Breton R.
      • Bélanger A.
      The key role of 17β-hydroxysteroid dehydrogenases in sex steroid biology.
      ,
      • Labrie F.
      • Luu-The V.
      • Labrie C.
      • Pelletier G.
      • El-Alfy M.
      Intracrinology and the skin.
      ;
      • Pelletier G.
      • Luu-The V.
      • Tetu B.
      • Labrie F.
      Immunocytochemical localization of type 5 17 beta-hydroxysteroid dehydrogenase in human reproductive tissues.
      ;
      • Qin K.N.
      • Rosenfield R.L.
      Expression of 17 beta-hydroxysteroid dehydrogenase type 5 in human ovary: a pilot study.
      ). 17β-HSD6, isolated by expression cloning of rat and human prostate, oxidizes 5α-androstane-3α, 17β-diol[3α-androstanediol (3α-Adiol)], to androsterone (
      • Biswas M.G.
      Russell DW: Expression cloning and characterization of oxidative 17beta- and 3alpha-hydroxysteroid dehydrogenases from rat and human prostate.
      ). It is a member of the short chain dehydrogenase/reductase family and shares 65% sequence identity with retinol dehydrogenase 1 (RoDH1), which catalyzes the oxidation of retinol to retinal. Expression of rat and human RoDH cDNA in mammalian cells is associated with the oxidative conversion of 3α-Adiol to 5α-DHT. Thus, 17β-HSD6 and RoDH play opposing roles in androgen action; 17β-HSD6 inactivates 3α-Adiol by conversion to androsterone and RoDH activates 3α-Adiol by conversion to 5α-DHT (
      • Biswas M.G.
      Russell DW: Expression cloning and characterization of oxidative 17beta- and 3alpha-hydroxysteroid dehydrogenases from rat and human prostate.
      ). The multisubstrate nature of these short chain dehydrogenase/reductase enzymes allows for retinoid/steroid interactions (
      • Napoli J.L.
      17beta-Hydroxysteroid dehydrogenase type 9 and other short-chain dehydrogenases/reductases that catalyze retinoid, 17beta- and 3alpha-hydroxysteroid metabolism.
      ). It is unknown if these interactions could partially explain the specific anti-proliferative effect of isotretinoin (13-cis retinoic acid) on sebocytes, whose growth and differentiation is strongly influenced by androgens (
      • Zouboulis ChC
      • Akamatsu H.
      • Stephanek K.
      • Orfanos C.E.
      Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaceous glands and their effect is antagonized by spironolactone.
      ;
      • Tsukada M.
      • Schröder M.
      • Roos T.C.
      • et al.
      13-cis retinoic acid exerts its specific activity on human sebocytes through selective intracellular isomerization to all-trans retinoic acid and binding to retinoid acid receptors.
      ). The human 17β-HSD7 shows 78% and 74% amino acid identity with rat and mouse 17β-HSD7, respectively. These enzymes are responsible for estradiol production in the corpus luteum during pregnancy, but are also present in placenta and several steroid target tissues (breast, testis, and prostate) as revealed by reverse transcription–polymerase chain reaction (
      • Krazeisen A.
      • Breitling R.
      • Imai K.
      • Fritz S.
      • Moller G.
      • Adamski J.
      Determination of cDNA, gene structure and chromosomal localization of the novel human 17beta-hydroxysteroid dehydrogenase type 7(1).
      ). Recently, 17β-HSD8, also known as Ke6 gene found in the HLA region, was shown to be able to transform efficiently estradiol to estrone in transfected HEK-293 cells (
      • Luu-The V.
      Analysis and characteristics of multiple types of human 17β-hydroxysteroid dehydrogenase.
      ). On the whole, the type 1, 3, 5, and 7 isoenzymes, using NADPH as cofactor, seem to reduce weak steroid hormones to more potent ones (e.g., DHEA into androstenediol, androstenedione to testosterone, 5α-androstanedione to 5α-DHT, estrone to estradiol), whereas type 2, 4, 6, and 8 isoenzymes, using NAD+ as cofactor, work in the opposite oxidizing direction and seem to play a general role in the peripheral inactivation of androgens (
      • Labrie F.
      • Luu-The V.
      • Lin S.X.
      • Labrie C.
      • Simard J.
      • Breton R.
      • Bélanger A.
      The key role of 17β-hydroxysteroid dehydrogenases in sex steroid biology.
      ;
      • Luu-The V.
      Analysis and characteristics of multiple types of human 17β-hydroxysteroid dehydrogenase.
      ). Only the 17β-HSD3, however, is responsible for pseudohermaphroditism in deficient boys.
      The cutaneous expression of 17β-HSD was mainly demonstrated in the pilosebaceous unit and epidermal keratinocytes. In hair follicles, 17β-HSD was histochemically localized to outer root sheath cells (ORS) (
      • Crovato F.
      • Moretti G.
      • Bertamino R.
      17β-hydroxysteroid dehydrogenases in hair follicles of normal and bald scalp: A histochemical study.
      ). The fresh plucked anagen hairs mainly containing keratinocytes from the inner root sheath and ORS expressed very high levels of 17β-HSD2 and moderate levels of 17β-HSD1 (
      • Courchay G.
      • Boyera N.
      • Bernard B.A.
      • Mahe Y.
      Messenger RNA expression of steroidogenesis enzyme subtypes in the human pilosebaceous unit.
      ). This is compatible with the early studies showing androstenedione as the major metabolite of cultured human hair follicle keratinocytes incubated with radiolabeled testosterone (
      • Dijkstra A.C.
      • Goos C.M.
      • Cunliffe W.J.
      • Sultan C.
      • Vermorken A.J.
      Is increased 5 alpha-reductase activity a primary phenomenon in androgen-dependent skin disorders.
      ;
      • Sonada T.
      • Itami S.
      • Kurata S.
      • Takayasu S.
      Testosterone metabolism by cultured human beard outer root sheath cells in comparison with epidermal keratinocytes.
      ). The human sebaceous gland possesses the cellular machinery needed to transcribe the genes for the type 1–5 isoenzymes of 17β-HSD (
      • Thiboutot D.
      • Martin P.
      • Volikos L.
      • Gilliland K.
      Oxidative activity of the type 2 isozyme of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) predominates in human sebaceous glands.
      ;
      • Labrie F.
      • Luu-The V.
      • Labrie C.
      • Pelletier G.
      • El-Alfy M.
      Intracrinology and the skin.
      ), among them a strong signal of 17β-HSD2 mRNA was detected (
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ). At the protein level, the type 2 isoenzyme of greater oxidative activity predominates in intact sebaceous glands, suggesting its protective role against the effects of excessive amounts of potent androgens in vivo (
      • Thiboutot D.
      • Martin P.
      • Volikos L.
      • Gilliland K.
      Oxidative activity of the type 2 isozyme of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) predominates in human sebaceous glands.
      ). Greater reductive activity of 17β-HSD was noted in sebaceous glands from facial areas compared with acne non-prone areas, suggesting an increased net production of potent androgens in facial areas (
      • Thiboutot D.
      • Martin P.
      • Volikos L.
      • Gilliland K.
      Oxidative activity of the type 2 isozyme of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) predominates in human sebaceous glands.
      ) and human sebocytes but not keratinocytes expressing 17β-HSD3, undersigning the major regulatory role of the sebaceous gland in androgen metabolism in the skin (
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ). The specific localization and importance of 17β-HSD2 in the physiology of sebaceous gland seems to be circumstantially evidenced by the description of “normal” development of pubertal acne and male distribution of body hair in male pseudohermaphroditism due to 17β-HSD3 deficiency syndrome (
      • Saez J.M.
      • de Peretti E.
      • Morera A.M.
      • David M.
      • Bertrand J.
      Familial male pseudohermaphroditism and gynecomastia due to a testicular 17-ketosteroid reductase defect. Studies in vivo.
      ;
      • Virdis R.
      • Saenger P.
      • Senior B.
      • New M.I.
      Endocrine studies in a pubertal male pseudohermaphrodite with 17-ketosteroid reductase deficiency.
      ). Noteworthy is the report of a distinct syndrome of alopecia totalis (actually atrichia), ichthyosis and male pseudohermaphroditism due to steroid 17β-HSD deficiency in an Israeli-Arab newborn infant (
      • Kauschansky A.
      • Shohat M.
      • Frydman M.
      • Rosler A.
      • Greenbaum E.
      • Sirota L.
      Syndrome of alopecia totalis and 17β-hydroxysteroid dehydrogenase deficiency.
      ).
      17β-HSD enzyme activity was also shown in cultured epidermal keratinocytes (
      • Itami S.
      • Takayasu S.
      Activity of 17 beta-hydroxysteroid dehydrogenase in various tissues of human skin.
      ;
      • Dijkstra A.C.
      • Goos C.M.
      • Cunliffe W.J.
      • Sultan C.
      • Vermorken A.J.
      Is increased 5 alpha-reductase activity a primary phenomenon in androgen-dependent skin disorders.
      ;
      • Hughes S.V.
      • Robinson E.
      • Bland R.
      • Lewis H.M.
      • Stewart P.M.
      • Hewison M.
      1,25-dihydroxyvitamin D3 regulates estrogen metabolism in cultured keratinocytes.
      ;
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ) and in the microdissected apocrine sweat gland (
      • Sonada T.
      • Itami S.
      • Kurata S.
      • Takayasu S.
      Testosterone metabolism by cultured human beard outer root sheath cells in comparison with epidermal keratinocytes.
      ). In primary cultured keratinocytes, which predominantly converted estradiol to estrone, mRNA expression of the type 1, 2, and 4 17β-HSD isoenzymes was detected and treatment with 1,25-dihydroxyvitamin D3 upregulated the type 2 mRNA (
      • Hughes S.V.
      • Robinson E.
      • Bland R.
      • Lewis H.M.
      • Stewart P.M.
      • Hewison M.
      1,25-dihydroxyvitamin D3 regulates estrogen metabolism in cultured keratinocytes.
      ). In HaCaT cells, a commonly used keratinocyte cell line, however, only the type 2 isoenzyme was detected (
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ).

      5α-REDUCTASE

      5α-reductase is the enzyme that catalyzes the conversion of testosterone to 5α-DHT. The conversion of testosterone to 5α-DHT amplifies the androgenic signal through two mechanisms: (i) 5α-DHT, unlike testosterone, cannot be aromatized to estrogen, thus its effect remains purely androgenic, and (ii) in vitro 5α-DHT binds to the human androgen receptor with greater affinity than testosterone does, and the 5α-DHT/androgen receptor complex appears to be more stable (
      • Anderson K.M.
      • Liao S.
      Selective retention of dihydrotestosterone by prostatic nuclei.
      ). Over the last decade, molecular cloning studies have characterized two genes that encode two isoenzymes of 5α-reductase, namely, type 1 and type 2; the former exists predominantly in the skin, whereas the latter in the prostate (
      • Andersson S.
      • Russell D.W.
      Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases.
      ;
      • Andersson S.
      • Berman D.M.
      • Jenkins E.P.
      • Russell D.W.
      Deletion of steroid 5α-reductase 2 gene in male pseudohermaphroditism.
      ). Genetically, the type 1 isoenzyme is encoded by the SRD5A1 gene on the distal arm of chromosome 5 (band p15), whereas the type 2 isoenzyme by the SRD5A2 gene on chromosome 2 (band p23) (
      • Russell D.W.
      • Wilson J.D.
      Steroid 5 alpha-reductase: two genes/two enzymes.
      ). Both genes contain five exons separated by four introns. Both isoenzymes are hydrophobic with approximately 50% identity in their amino acid sequences. Whereas type 1 5α-reductase has a broad alkaline pH optima of 6.0–8.5 and demonstrates relatively moderate affinity for steroid substrates (Km: 1–5 μM), the type 2 5α-reductase has a narrow acidic pH optima of 5.0–6.0 and demonstrates high affinity for substrates (Km: 4–50 nm) (
      • Russell D.W.
      • Wilson J.D.
      Steroid 5 alpha-reductase: two genes/two enzymes.
      ). These different enzyme kinetics may have some important implications for disease states. Cutaneous distribution of type 1 isoenzyme in vivo was immunohistochemically identified in sebaceous glands, epidermis, eccrine sweat glands, apocrine sweat glands (in normal ones as well as in people with osmidrosis), and hair follicles (ORS, DP, matrix), as well as in the endothelial cells of small vessels and the Schwann cells of cutaneous myelinated nerves (
      • Luu-The V.
      • Sugimoto Y.
      • Puy L.
      • Labrie Y.
      • Solache I.L.
      • Singh M.
      • Labrie F.
      Characterization, expression, and immunohistochemical localization of 5α-reductase in human skin.
      ;
      • Eicheler W.
      • Dreher M.
      • Hoffmann R.
      • Happle R.
      • Aumüller G.
      Immunohistochemical evidence for differential distribution of 5α-reductase isozymes in human skin.
      ;
      • Courchay G.
      • Boyera N.
      • Bernard B.A.
      • Mahe Y.
      Messenger RNA expression of steroidogenesis enzyme subtypes in the human pilosebaceous unit.
      ;
      • Sato T.
      • Sonada T.
      • Itami S.
      • Takayasu S.
      Predominance of type 1 5 alpha-reductase in apocrine sweat glands of patients with excessive or abnormal odour derived from apocrine gland (osmidrosis).
      ). In the skin the activity of the type 1 5α-reductase is concentrated in sebaceous glands and is significantly higher in sebaceous glands from the face and scalp compared with nonacne-prone areas (
      • Thiboutot D.
      • Harris G.
      • Iles V.
      • Cimis G.
      • Gilliland K.
      • Hagari S.
      Activity of the type 1 5 alpha-reductase exhibits regional differences in isolated sebaceous glands and whole skin.
      ). In vitro, type 1 5α-reductase was detected in the cytoplasm of cultured human sebocytes, keratinocytes and HaCaT cells, fibroblasts, dermal microvascular endothelial cells, hair DP cells from various body sites, melanocytes, and melanoma cells (
      • Chen W.
      • Zouboulis C.C.
      • Fritsch M.
      • et al.
      Evidence of heterogeneity and quantitative differences of the type 1 5alpha-reductase expression in cultured human skin cells—evidence of its presence in melanocytes.
      ,
      • Chen W.
      • Zouboulis C.C.
      • Fritsch M.
      • Kodelja V.
      • Orfanos C.E.
      Heterogeneity and quantitative differences of type 1 5 alpha-reductase expression in cultured skin epithelial cells.
      ;
      • Ando Y.
      • Yamaguchi Y.
      • Hamada K.
      • Yoshikawa K.
      • Itami S.
      Expression of mRNA for androgen receptor, 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase in human dermal papilla cells.
      ;
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ). Northern blot studies revealed most abundant type 1 mRNA in neonatal foreskin keratinocytes, followed by adult facial sebocytes, and stronger expression in DP from occipital hair cells than from beard (
      • Chen W.
      • Zouboulis C.C.
      • Fritsch M.
      • et al.
      Evidence of heterogeneity and quantitative differences of the type 1 5alpha-reductase expression in cultured human skin cells—evidence of its presence in melanocytes.
      ).
      Within hair follicles, prominent immunostaining of type 2 5α-reductase was localized in the inner layer of ORS, inner root sheath, the infundibulum of hair follicle, and sebaceous ducts (
      • Bayne E.K.
      • Flanagan J.
      • Einstein M.
      • et al.
      Immunohistochemical localization of types 1 and 2 5 alpha-reductase in human scalp.
      ). Regional studies showed the type 2 mRNA present in beard DP, but absent from occipital scalp and axillary DP (
      • Ando Y.
      • Yamaguchi Y.
      • Hamada K.
      • Yoshikawa K.
      • Itami S.
      Expression of mRNA for androgen receptor, 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase in human dermal papilla cells.
      ). The type 2 isoenzyme in beard DP has three times higher activity than the type 1 5α-reductase present in the occipital scalp and axillary DP (
      • Itami S.
      • Sonada T.
      • Kurata S.
      • Takayasu S.
      Mechanism of action of androgen in hair follicles.
      ;
      • Eicheler W.
      • Happle R.
      • Hoffmann R.
      5 alpha-reductase activity in the human hair follicle concentrates in the dermal papilla.
      ). The specific activity of 5α-reductase in the hair DP exceeded those in other hair follicle compartments (connective tissue sheaths and ORS) by a factor of at least 14 in the scalp and at least 80 in the beard (
      • Eicheler W.
      • Happle R.
      • Hoffmann R.
      5 alpha-reductase activity in the human hair follicle concentrates in the dermal papilla.
      ). The beard DP cells appeared to generate more 5α-DHT than those from nonbalding scalp hair follicles (
      • Thornton M.J.
      • Laing I.
      • Hamada K.
      • Messenger A.G.
      • Randall V.A.
      Differences in testosterone metabolism by beard and scalp hair follicle dermal papilla cells.
      ); however, the individual freshly isolated intact DP was shown to possess considerably different levels of ex vivo enzyme activities (
      • Niiyama S.
      • Kojima K.
      • Hamada T.
      • Happle R.
      • Hoffmann R.
      The novel drug CS-891 inhibits 5 alpha-reductase activity in freshly isolated dermal papilla of human hair follicles.
      ).
      Taken together, whereas the type 1 5α-reductase has been definitely demonstrated in sebaceous glands, the isoenzyme distri-bution in hair follicles is less well-defined, probably due to: (i) physiologic variation of the enzyme activity in different body regions; (ii) utilization of different polyclonal/monoclonal antibodies; and (iii) inadequate assessment of enough specimens. More evidence is needed to define better the existence of the type 1 isoenzyme in hair follicles (
      • Luu-The V.
      • Sugimoto Y.
      • Puy L.
      • Labrie Y.
      • Solache I.L.
      • Singh M.
      • Labrie F.
      Characterization, expression, and immunohistochemical localization of 5α-reductase in human skin.
      ;
      • Eicheler W.
      • Dreher M.
      • Hoffmann R.
      • Happle R.
      • Aumüller G.
      Immunohistochemical evidence for differential distribution of 5α-reductase isozymes in human skin.
      ;
      • Chen W.
      • Zouboulis C.C.
      • Fritsch M.
      • et al.
      Evidence of heterogeneity and quantitative differences of the type 1 5alpha-reductase expression in cultured human skin cells—evidence of its presence in melanocytes.
      ;
      • Ando Y.
      • Yamaguchi Y.
      • Hamada K.
      • Yoshikawa K.
      • Itami S.
      Expression of mRNA for androgen receptor, 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase in human dermal papilla cells.
      ;
      • Bayne E.K.
      • Flanagan J.
      • Einstein M.
      • et al.
      Immunohistochemical localization of types 1 and 2 5 alpha-reductase in human scalp.
      ) and the precise localization of the type 2 isoenzyme within the hair follicles (in ORS or in DP) (
      • Hoffmann R.
      • Happle R.
      Finasteride is the main inhibitor of 5 alpha-reductase activity in microdissected dermal papillae of human hair follicles.
      ;
      • Bayne E.K.
      • Flanagan J.
      • Einstein M.
      • et al.
      Immunohistochemical localization of types 1 and 2 5 alpha-reductase in human scalp.
      ).

      CYTOCHROME P450 19 (AROMATASE)

      Aromatase, the product of the CYP19 gene, catalyzes three consecutive hydroxylation reactions converting C19 androgens to C18 estrogens. It belongs to the cytochrome P450 superfamily of enzymes, which contains 36 gene families and over 300 characterized members (
      • Simpson E.R.
      • Zhao Y.
      • Agarwal V.R.
      • et al.
      Aromatase expression in health and disease.
      ). Recently, aromatase has been reported to be present in various extragonadal tissues and its expression is regulated in part by means of tissue-specific promoters through the alternative splicing mechanism on multiple exons 1 (
      • Simpson E.R.
      Role of aromatase in sex steroid action.
      ). At least six variants of exon 1 have been described; exons 1a, 1b, 1c, 1d, 1e, and 1f that are specific for expression in the placenta, skin fibroblasts/fetal liver/adipose tissue/vascular tissue, ovary, ovary/prostate, placenta, and fetal brain, respectively (
      • Harada N.
      [A new aspect of the pharmacological and physiological significance of the aromatase/estrogen system]. [Japanese].
      ,
      • Harada N.
      Aromatase and intracrinology of estrogen in hormone-dependent tumors.
      ). By immunohistochemical examination, aromastase was found in the ORS of anagen, terminal hair follicles, and in sebaceous glands, but rarely in telogen hair follicles. The expression did not vary with body site or sex in normal subjects, but seemed to differ in patients with androgenetic alopecia (
      • Sawaya M.E.
      • Penneys N.S.
      Immunohistochemical distribution of aromatase and 3 β-hydroxysteroid dehydrogenase in human hair follicle and sebaceous gland.
      ;
      • Sawaya M.E.
      • Price V.H.
      Different levels of 5 alpha-reductase type I and II, aromatase, and androgen receptor in hair follicles of women and men with androgenetic alopecia.
      ; see later). Semiquantitative reverse transcription–polymerase chain reaction methods, however, showed poor expression of aromatase in the plucked hair containing ORS and inner root sheath keratinocytes (
      • Courchay G.
      • Boyera N.
      • Bernard B.A.
      • Mahe Y.
      Messenger RNA expression of steroidogenesis enzyme subtypes in the human pilosebaceous unit.
      ). The aromatase enzyme activity has also been demonstrated in keratinocytes cultured in serum-free medium (
      • Hughes S.V.
      • Robinson E.
      • Bland R.
      • Lewis H.M.
      • Stewart P.M.
      • Hewison M.
      1,25-dihydroxyvitamin D3 regulates estrogen metabolism in cultured keratinocytes.
      ), fibroblasts from both genital and nongenital skin (
      • Svenstrup B.
      • Brunner N.
      • Dombernowsky P.
      • Nohr I.
      • Micic S.
      • Bennett P.
      • Spang-Thomsen M.
      Comparison of the effect of cortisol on aromatase activity and androgen metabolism in two human fibroblast cell lines derived from the same individual.
      ), and fibroblasts from adipose tissue (
      • Rink J.D.
      • Simpson E.R.
      • Barnard J.J.
      • Bulun S.E.
      Cellular characterization of adipose tissue from various body sites of women.
      ). In most tissues, aromatase is induced by cyclic adenosine monophosphate or factors utilizing cyclic adenosine monophosphate as a second messenger, whereas androgens and glucocorticoids have been shown to be able to stimulate the expression or activity of aromatase (
      • Stillman S.C.
      • Evans B.A.
      • Hughes I.A.
      Androgen dependent stimulation of aromatase activity in genital skin fibroblasts from normals and patients with androgen insensitivity.
      ;
      • Zhao Y.
      • Mendelson C.R.
      • Simpson E.R.
      Characterization of the sequences of the human CYP 19 (aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes.
      ;
      • Harada N.
      Aromatase and intracrinology of estrogen in hormone-dependent tumors.
      ). To understand better the clinical significance of aromatase in androgen-dependent dermatoses, it would be interesting to study patients affected with aromatase deficiency or excess syndromes to observe the occurrence of acne or androgenetic alopecia (
      • Simpson E.R.
      Genetic mutations resulting in estrogen insufficiency in the male.
      ,
      • Simpson E.R.
      Genetic mutations resulting in loss of aromatase activity in humans and mice.
      ;
      • Bulun S.E.
      • Noble L.S.
      • Takayama K.
      • et al.
      Endocrine disorders associated with inappropriately high aromatase expression.
      ;
      • Bulun S.E.
      Aromatase deficiency and estrogen resistance: from molecular genetics to clinic.
      ).

      3α-HSD

      Mammalian 3α-HSD regulate steroid hormone levels. Hepatic 3α-HSD inactivate circulating androgens, progestins, and glucocorticoids by catalyzing the conversion of 3-ketosteroids to 3α-hydroxy compounds, e.g., the transformation of 5α-DHT into 3α-Adiol. In the prostate, it acts as a molecular switch and controls the amount of 5α-DHT that can bind to the androgen receptor, whereas in the brain 3α-HSD can regulate the amount of tetrahydrosteroids that can alter γ-aminobutyric acid receptor function (
      • Penning T.M.
      • Pawlowski J.E.
      • Schlegel B.P.
      • et al.
      Mannalian 3 alpha-hydroxysteroid dehydrogenases.
      ). Molecular cloning indicates that these mammalian 3α-HSD are highly homologous proteins, prefer NADPH as cofactors and belong to the aldo-keto reductase superfamily, including also 17β-HSD5, ovarian 20α-HSD as well as the steroid 5β-reductases (
      • Dufort I.
      • Soucy P.
      • Labrie F.
      • Luu-The V.
      Molecular cloning of human type 3 3alpha-hydroxysteroid dehydrogenase that differs from 20 alpha-hydroxysteroid dehydrogeanse by seven amino acids.
      ;
      • Penning T.M.
      Molecular determinants of steroid recognition and catalysis in aldoketo reductases. Lessons from 3 alpha-hydroxysteroid dehydrogenase.
      ). The human type 1 and type 3 isoenzymes, sharing 81.7% identity, both efficiently catalyze the transformation of 5β-DHT into 3α-Adiol in intact vector-transfected transformed human embryonic kidney cells (
      • Dufort I.
      • Soucy P.
      • Labrie F.
      • Luu-The V.
      Molecular cloning of human type 3 3alpha-hydroxysteroid dehydrogenase that differs from 20 alpha-hydroxysteroid dehydrogeanse by seven amino acids.
      ,
      • Dufort I.
      • Labrie F.
      • Luu-The V.
      Human types 1 and 3 3alpha-hydroxysteroid dehydrogenase: differential lability and tissue distribution.
      ). The human type 1 3α-HSD is expressed exclusively in the liver, whereas the type 3 is more widely expressed in the liver, adrenal, testis, brain, prostate, and HaCaT keratinocytes. The expression of the type 2 3α-HSD was shown in the human prostate, and there it inactivated 5α-DHT through its 3-ketosteroid reductase activity (
      • Lin H.K.
      • Jez J.M.
      • Schlegel B.P.
      • Peehl D.M.
      • Pachter J.A.
      • Penning T.M.
      Expression and characterization of recombinant type 2 3 alpha-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3 alpha/17 beta-HSD activity and cellular distribution.
      ).
      Early studies showed the enzyme activity of 3α-HSD in cultured human skin fibroblasts and the 5α-DHT reduction is three times higher in fibroblasts from genital areas than from nongenital areas (
      • Sultan C.
      • Coupe M.
      • Devillier C.
      • Chavis C.
      • Terraza A.
      • Descomps B.
      Metabolism of dihydrotestosterone in cultured skin fibroblasts: reduction to 5 alpha-androstane-3-alpha, 17 beta-diol.
      ). The expression of 3α-HSD mRNA has been demonstrated in sebocytes (SZ95), keratinocytes (HaCaT cells), and melanoma cells (MeWo), but the isotype has not yet been identified (
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ). Keratinocytes were found to inactivate tissue active androgens in a more pronounced manner than sebocytes do by engagement of 3α-HSD (
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ). Clinically, it is still controversial if serum levels of 3α-Adiol conjugates (3α-Adiol glucuronide or 3α-Adiol sulfate) serve as reliable indicator for cutaneous 5α-DHT formation (see later,
      • Lookingbill D.P.
      • Demers L.M.
      • Tigelaar R.E.
      • Shalita A.R.
      Effect of isotretinoin on serum levels of precursor and peripherally derived androgens in patients with acne.
      ;
      • Horton R.
      Dihydrotestosterone is a peripheral paracrine hormone.
      ;
      • Vogt C.
      • Dericks-Tan J.S.
      • Kuhl H.
      • Taubert H.D.
      Is 3 alpha, 17 beta-androstanediol-glucuronide a diagnostic marker in women with androgenic manifestations?.
      ), or are just a marker of adrenal steroid production and metabolism (
      • Rittmaster R.S.
      Clinical relevance of testosterone and dihydrotestosterone metabolism in women.
      ). Interestingly, rat liver 3α-HSD was found to be a target for nonsteroidal anti-inflammatory drugs (
      • Pawlowski J.
      • Huizinga M.
      • Penning T.M.
      Isolation and partial characterization of a full-length cDNA clone for 3 alpha-hydroxysteroid dehydrogenase: a potential target enzyme for nonsteroidal anti-inflammatory drugs.
      ).
      Cutaneous distribution of steroidogenic isozymes is summarized in Table I.
      Table ICutaneous distribution of steroidogenic isozymes
      Isoenzymes/major cutaneous isoenzymeEncoding genes of the cutaneous isoenzymeCutaneous distribution
      Steroid sulfatase?chromosome Xp22K, F, DP
      3β-HSDTypes 1 and 2/Type 1chromosome 1p13.1SG
      17β-HSDTypes 1-7/Type 2chromosome 16q24SG, K, A
      5α-reductaseTypes 1-2/Type 1chromosome 5p15SG, K, F, En, M, A, Ec, DP, ORS
      Aromatase?chromosome 15q21SG, ORS, K, F
      3α-HSDTypes 1-3/Type 3?chromosome 10p15SG, K, M, F
      ? Not known, HSD: hydroxysteroid dehydrogenase, K: keratinocytes, F: fibroblast, DP: hair dermal papilla, SG: sebaceous glands, A: apocrine sweat gland, Ec: eccrine sweat gland, En: vascular endothelial cells, M: melanocytes, ORS: outer root sheath

      ANDROGEN-DEPENDENT DERMATOSES

      Acne vulgaris

      Decades of investigation have firmly established that the development and secretory activity of sebaceous glands are strikingly influenced by hormones, especially androgens, and that the sebaceous glands at the same time dominates cutaneous androgen production (
      • Pochi P.E.
      • Strauss J.S.
      Endocrinologic control of the development and activity of the human sebaceous gland.
      ;
      • Zouboulis ChC
      • Xia L.
      • Akamatsu H.
      • et al.
      The human sebocyte culture model provides new insights into development and management of seborrhoea and acne.
      ,2002;
      • Zouboulis ChC
      Human skin: An independent peripheral endocrine organ.
      ). The distribution of various hydroxysteroid dehydrogenases in human sebaceous glands and their strong activities in acne-prone skin in comparison with nonacne-prone skin areas have long since been evaluated (
      • Baillie A.H.
      • Thomson J.
      • Milne J.A.
      The distribution of hydroxysteroid dehydrogenase in human sebaceous glands.
      ).
      Strong steroid sulfatase immunoreactivity in the acne skin, primarily associated with the monocytes infiltrating the lesions, but not in unaffected skin was observed. The enzymatic hydrolysis of DHEA-S to DHEA and of estrone sulfate to estrone in cultured epidermal keratinocytes has been demonstrated (
      • Milewich L.
      • Sontheimer R.D.
      • Herndon Jr, J.H.
      Steroid sulfatase activity in epidermis of acne-prone and non-acne-prone skin of patients with acne vulgaris.
      ). There were no differences in the rates of enzymatic hydrolysis of steroid sulfatase in the epidermis of acne-prone and nonacne-prone skin; however, the rate of estrone sulfate hydrolysis was two to eight times greater than that of DHEA-S in all of the tissues evaluated.
      Significant differences in the activity of 5α-reductase or 17β-HSD in sebaceous glands regarding the presence of acne were neither noted in men nor in women (
      • Thiboutot D.
      • Gilliland K.
      • Light J.
      • Lookingbill D.
      Androgen metabolism in sebaceous glands from subjects with and without acne.
      ). The activity of 5α-reductase or 17β-HSD was significantly greater in sebaceous glands from men than women. Higher serum androgen levels were significantly higher in women with acne, whereas no differences were noted in men on the basis of the presence of acne.
      The exclusive predominance of the type 1 5α-reductase in sebaceous glands and the major influence of local androgenesis on sebum production were further confirmed by two clinical observations: (i) adult males with type 2 5α-reductase deficiency had sebum production scores identical to normal age-matched males; (ii) males with benign prostate hyperplasia treated with finasteride (5 mg per day for 1 y) did not decrease the sebum score from baseline values, although the serum 5α-DHT level was lowered (
      • Imperato-McGinley J.
      • Gautier T.
      • Cai L.Q.
      • Yee B.
      • Epstein J.
      • Pochi P.
      The androgen control of sebum production. Studies of subjects with dihydrotestosterone deficiency and complete androgen insensitivity.
      ). Moreover, the activity of the type 1 5α-reductase exhibits regional differences in isolated sebaceous glands (
      • Thiboutot D.
      • Harris G.
      • Iles V.
      • Cimis G.
      • Gilliland K.
      • Hagari S.
      Activity of the type 1 5 alpha-reductase exhibits regional differences in isolated sebaceous glands and whole skin.
      ), which seemed to correlate with the finding that the stimulatory effect of 5α-DHT on cell proliferation, was more prominent on facial than on nonfacial sebocytes (
      • Akamatsu H.
      • Zouboulis C.C.
      • Orfanos C.E.
      Control of human sebocyte proliferation in vitro by testosterone and 5-alpha-dihydrotestosterone is dependent on the localization of the sebaceous glands.
      ). In normal hair follicles and in open and closed comedones, the type 2 isoenzyme was demonstrated to localize within the companion layer of the follicle (innermost layer of the ORS). In inflammatory acne lesions, the type 2 isozyme localized to the companion layer of hair follicles and endothelial cells within the surrounding inflammatory infiltrate, but not to sebaceous glands (
      • Thiboutot D.
      • Bayne E.
      • Thorne J.
      • et al.
      Immunolocalization of 5 alpha-reductase isozymes in acne lesions and normal skin.
      ). As 5α-DHT could enhance 5α-reductase mRNA and enzyme activity in a feed-forward regulation (
      • Russell D.W.
      • Wilson J.D.
      Steroid 5 alpha-reductase: two genes/two enzymes.
      ), and the type 2 isoenzyme has higher affinity for testosterone than the type 1 isoenzyme, the up-regulated type 2 isoenzyme in the diseased state might contribute to the aggravation of the pre-existing “hyperandrogenic” condition.
      The role of 3α-HSD in the pathogenesis of acne has rarely been addressed; the elevated plasma level of 3α-Adiol glucuro-nide might merely reflect the increased production of 5α-DHT, decreased local enzyme activity of 3α-HSD to metabolize 5α-DHT, or both. It is not known if different levels of 3α-HSD occur between men and women, between acne and nonacne subjects or between mild (papulopustular) acne and severe (nodulocytic) acne patients. In women with mild to moderate acne, plasma 3α-Adiol glucuronide was suggested to be the most sensitive marker (
      • Lookingbill D.P.
      • Horton R.
      • Demers L.M.
      • Egan N.
      • Marks Jr, J.G.
      • Santen R.J.
      Tissue production of androgens in women with acne.
      ), whereas the levels were shown to be decreased or within normal range in other studies (
      • Toscano V.
      • Balducci R.
      • Bianchi P.
      • et al.
      Two different pathogenetic mechanisms may play a role in acne and in hirsutism.
      ;
      • Joura E.A.
      • Geusau A.
      • Schneider B.
      • Soregi G.
      • Huber J.C.
      Serum 3alpha-androstanediol-glucuronide is decreased in nonhirsute women with acne vulgaris.
      ). Exaggerated androsterone metabolism, however, was observed in hype-randrogenic as well as in some normo-androgenic women with acne (
      • Carmina E.
      • Stanczyk F.Z.
      • Matteri R.K.
      • Lobo R.A.
      Serum androsterone conjugates differentiate between acne and hirsutism in hyperandrogenic women.
      ;
      • Carmina E.
      • Lobo R.A.
      Evidence for increased androsterone metabolism in some normoandrogenic women with acne.
      ), and androsterone glucuronide/sulfate seemed to be a better marker than 3α-Adiol glucuronide in differentiating acne and hirsutism in hyperandrogenic women (
      • Carmina E.
      • Stanczyk F.Z.
      • Matteri R.K.
      • Lobo R.A.
      Serum androsterone conjugates differentiate between acne and hirsutism in hyperandrogenic women.
      ). In young men, a statistically significant correlation was found between serum 3α-Adiol glucuronide and chest hairiness, acne as well as a combined chest hairiness and acne score (
      • Lookingbill D.P.
      • Egan N.
      • Santen R.J.
      • Demers L.M.
      Correlation of serum 3 alpha-androstanediol glucuronide with acne and chest hair density in men.
      ). Noteworthy was the decreased serum levels of 3α-Adiol glucuronide in 24 acne subjects (15 men and nine women) treated with 1 mg isotretinoin per kg bodyweight per day for 20 wk (
      • Lookingbill D.P.
      • Demers L.M.
      • Tigelaar R.E.
      • Shalita A.R.
      Effect of isotretinoin on serum levels of precursor and peripherally derived androgens in patients with acne.
      ).
      It is also interesting to note that MPV-2213, a novel nonsteroidal competitive inhibitor of aromatase, could lead to the adverse effect of acne formation in healthy male subjects (
      • Sahokoski O.
      • Irjala K.
      • Huupponen R.
      • Halonen K.
      • Salminen E.
      • Scheinin H.
      Hormonal effects of MPV-2213ad, a new selective aromatase inhibitor, in healthy male subjects. A phase I study.
      ), which sheds light on the possible role of aromatase in the pathophysiology of acne formation.

      Androgenetic alopecia

      Androgenetic alopecia can be defined as a 5α-DHT-dependent process with continuous miniaturization of androgen sensitive hair follicles (
      • Hoffmann R.
      • Happle R.
      Current understanding of androgenetic alopecia: Part I: etiopathogenesis.
      ). The intrafollicular conversion of testosterone to 5α-DHT seems to play a central part leading to androgenetic hair loss.
      Because steroid sulfatase plays an important part in androgen metabolism, and elevated levels of DHEA have been reported in young men with androgenetic alopecia, the hypothesis was advanced that men with X-linked recessive ichthyosis do not show androgenetic alopecia or develop only mild forms of common baldness (
      • Happle R.
      • Hoffmann R.
      Absence of male-pattern baldness in men with X-linked recessive ichthyosis? A hypothesis to be challenged.
      ). A recent clinical survey, however, did not support this hypothesis that X-linked recessive ichthyosis and androgenetic alopecia are mutually exclusive, in as much as advanced androgenetic alopecia was found among these men (
      • Trueb R.M.
      Meyer JC: Male-pattern baldness in men with X-linked recessive ichthyosis.
      ).
      Homogenates of sebaceous glands from a balding scalp had greater 3β-HSD activity than those from a hairy scalp (
      • Sawaya M.E.
      • Honig L.S.
      • Garland L.D.
      • Hsia S.L.
      Delta 5-3-beta-hydroxysteroid dehydrogenase activity in sebaceous glands of scalp in male-pattern baldness.
      ). There is no data about the activity of 3β-HSD in hair follicles from hairy vs balding scalp. Isolated intact hair follicles and sebaceous glands from balding frontal scalp, compared with a nonbalding occipital scalp, of patients with androgenetic alopecia demonstrated increased activity of 17β-HSD (
      • Sawaya M.E.
      Steroid chemistry and hormone controls during the hair follicle cycle.
      ).
      Both women and men with androgenetic alopecia have higher levels of androgen receptors and 5α-reductase type 1 and 2 in frontal than in occipital hair follicles, whereas higher levels of aromatase were found in their occipital follicles (
      • Sawaya M.E.
      • Price V.H.
      Different levels of 5 alpha-reductase type I and II, aromatase, and androgen receptor in hair follicles of women and men with androgenetic alopecia.
      ). Aromatase content in women's frontal hair follicles was six times greater than in frontal hair follicles in men. Frontal hair follicles in women had 3 and 3.5 times less 5α-reductase type 1 and 2, respectively, than frontal hair follicles in men (
      • Sawaya M.E.
      • Price V.H.
      Different levels of 5 alpha-reductase type I and II, aromatase, and androgen receptor in hair follicles of women and men with androgenetic alopecia.
      ). Finasteride, a specific competitive inhibitor of type 2 5α-reductase, maximally decreased both scalp skin and serum 5α-DHT at doses as low as 0.2 mg per day (
      • Drake L.
      • Hordinsky M.
      • Fiedler V.
      • et al.
      The effects of finasteride on scalp skin and serum androgen levels in men with androgenetic alopecia.
      ). Minoxidil was lately shown to increase 17β-HSD and 5α-reductase activity of cultured human DP from balding scalp (
      • Sato T.
      • Tadokoro T.
      • Sonoda T.
      • Asada Y.
      • Itami S.
      • Takayasu S.
      Minoxidil increases 17β-HSD and 5α-reductase activity of cultured human dermal papilla cells from balding scalp.
      ), the relevant significance of which remains unclear.
      A disorder of androgen conjugation, favoring sulfurylation over glucuronidation, has been suggested to be a characteristic feature in men and women with androgenetic alopecia (
      • Legro R.S.
      • Carmina E.
      • Stanczyk F.Z.
      • Gentzschein E.
      • Lobo R.A.
      Alterations in androgen conjugate levels in women and men with alopecia.
      ). Women with female pattern baldness were noted to have a marked increase in the 3α-Adiol glucuronide/sex hormone binding globulin ratio and low serum level of sex hormone binding globulin (
      • de Villez R.L.
      • Dunn J.
      Female androgenic alopecia. The 3 alpha, 17 beta-androstanediol glucuronide/sex hormone binding globulin ratio as a possible marker for female pattern baldness.
      ).
      In summary, the current understanding of androgenetic alopecia focuses on the excessive in situ conversion of testo-sterone to 5α-DHT, which involves the hyperactivity of 5α-reductase and 17β-HSD, coupled with hypoactivity of aromatase and overexpression of androgen receptors in balding vs nonbalding scalps (
      • Kaufman K.D.
      Androgen metabolism as it affects hair growth in androgenetic alopecia.
      ). 5α-DHT was currently shown to induce apoptosis in DP in vitro via a bcl-2 related pathway.
      Wróbel A, Mandt N, Hossini A, Seltmann H, Zouboulis ChC, Orfanos CE, Blume-Peytavi U: 5α-Dihydrotestosterone and testosterone induce apoptosis in human dermal papilla cells by downregulation of the bcl-2 pathway. J Invest Dermatol 115:581, 2000 (Abstr.)
      The roles of steroid sulfatase, 3β-Δ5-HSD and 3α-HSD merit further studies. The molecular steps involved in androgen-dependent beard growth vs androgen-dependent hair miniaturization in androgenetic alopecia remain obscure (
      • Hoffmann R.
      • Happle R.
      Current understanding of androgenetic alopecia: Part I: etiopathogenesis.
      ). The newly emerging interest in the function of sebaceous glands in follicular biology (
      • Sundberg J.P.
      • Boggess D.
      • Sundberg B.A.
      • Eilertsen K.
      • Parimoo S.
      • Filippi M.
      • Stenn K.
      Asebia-2J (Scd1(ab2J)): a new allele and a model for scarring alopecia.
      ; Zouboulis et al, 2002) may arouse the re-evaluation of the role of sebaceous glands in the peripheral hyperandrogenism occurring in androgenetic alopecia (
      • Dijkstra A.C.
      • Goos C.M.
      • Cunliffe W.J.
      • Sultan C.
      • Vermorken A.J.
      Is increased 5 alpha-reductase activity a primary phenomenon in androgen-dependent skin disorders.
      ;
      • Sawaya M.E.
      • Honig L.S.
      • Garland L.D.
      • Hsia S.L.
      Delta 5-3-beta-hydroxysteroid dehydrogenase activity in sebaceous glands of scalp in male-pattern baldness.
      ;
      • Zouboulis ChC
      • Akamatsu H.
      • Stephanek K.
      • Orfanos C.E.
      Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaceous glands and their effect is antagonized by spironolactone.
      ).

      HIRSUTISM

      The prevalence of hirsuties is difficult to assess. Earlier studies estimated hirsutism affects between 5 and 10% of women (
      • Azziz R.
      • Carmina E.
      • Sawaya M.E.
      Idiopathic hirsutism.
      ). Racial as well as social factors, and nowadays the media, however, greatly determine the threshold level for normality of hair growth (
      • Dawber R.P.
      • Sinclair R.D.
      Hirsuties.
      ). Hirsutism is often seen in endocrine disorders characterized by hyperandrogenesis as the result of abnormalities of either the ovaries or adrenal glands (
      • Dawber R.P.
      • Sinclair R.D.
      Hirsuties.
      ). The diagnosis of idiopathic hirsutism, when strictly defined as hirsuteness with normal ovulatory function and circulating androgen levels, will include less than 20% of all hirsute women (
      • Azziz R.
      • Carmina E.
      • Sawaya M.E.
      Idiopathic hirsutism.
      ). Earlier studies showed in adult women the higher activity of 5α-reductase in the genital skin as compared with the dorsal skin of the fetus as well as the abdominal skin, respectively (
      • Flamigni C.
      • Collins W.P.
      • Koullapis E.N.
      • Craft I.
      • Sommerville I.F.
      Androgen metabolism in human skin.
      ), and in hirsute women the increased 5α-reductase activity in genital skin (
      • Jenkins J.S.
      • Ash S.
      The metabolism of testosterone by human skin in disorders of hair growth.
      ;
      • Serafini P.
      • Lobo R.A.
      Increased 5 alpha-reductase activity in idiopathic hirsutism.
      ;
      • Serafini P.
      • Ablan F.
      • Lobo R.A.
      5 alpha-reductase activity in the genital skin of hirsute women.
      ). Expression of both type 1 and type 2 5α-reductase mRNA was demonstrated in genital skin as well as in pubic skin fibroblasts, whereas the type 2 isoenzyme appeared to predominate in pubic skin of normal men, normal women, and hirsute patients (
      • Mestayer C.
      • Berthaut I.
      • Portois M.C.
      • Wright F.
      • Kuttenn F.
      • Mowszowicz I.
      • Mauvais-Jarvis P.
      Predominant expression of 5 alpha-reductase type 1 in pubic skin from normal subjects and hirsute patients.
      ). The effectiveness of finasteride (5 mg per day) in the treatment of idiopathic and polycystic ovary syndrome-associated hirsutism further signifies the role of type 2 5α-reductase in the pathophysiology of hirsutism (
      • Petrone A.
      • Civitillo R.M.
      • Galante L.
      • Giannotti F.
      • D'Anto V.
      • Rippa G.
      • Tolino A.
      Usefulness of a 12-month treatment with finasteride in idiopathic and polycystic ovary syndrome-associated hirsutism.
      ). As the circulating androgens are known to increase peripheral 5α-reductase activity (
      • Azziz R.
      • Carmina E.
      • Sawaya M.E.
      Idiopathic hirsutism.
      ), and as previous studies mostly utilized whole skin tissue or cultured fibroblasts, but not the individual hair follicle components, however, more work is needed to define better the precise roles of the different isoenzymes and their activities in the development of hirsutism, especially the idiopathic hirsutism.
      3α-Adiol conjugates (glucuronide or sulfate) were elevated in hirsute compared with nonhirsute women (
      • Carmina E.
      • Stanczyk F.Z.
      • Matteri R.K.
      • Lobo R.A.
      Serum androsterone conjugates differentiate between acne and hirsutism in hyperandrogenic women.
      ;
      • Toscano V.
      • Balducci R.
      • Bianchi P.
      • et al.
      Two different pathogenetic mechanisms may play a role in acne and in hirsutism.
      ). Plasma (serum) concentrations of 3α-Adiol glucuronide appeared to reflect hirsutism most accurately (
      • Carmina E.
      • Gentzschein E.
      • Stanczyk F.Z.
      • Lobo R.A.
      Substrate dependency of C19 conjugates in hirsute hyperandrogenic women and the influence of adrenal androgen.
      ) and were elevated in polycystic ovary syndrome patients with/without hirsutism and in patients with idiopathic hirsutism (
      • Kirschner M.A.
      • Samojlik E.
      • Szmal E.
      Clinical usefulness of plasma androstanediol glucuronide measurements in women with idiopathic hirsutism.
      ;
      • Falsetti L.
      • Rosina B.
      • de Fusco D.
      Serum levels of 3 alpha-androstanediol glucuronide in hirsute and non-hirsute women.
      ). In idiopathic hirsutism, the specificity of serum 3α-Adiol glucuronide in reflecting the peripheral 5α-reductase activity has been questioned (
      • Vermeulen A.
      • Giagulli V.A.
      Physiology of plasma androstanediol glucuronide.
      ;
      • Gilad S.
      • Chayen R.
      • Tordjman K.
      • Kisch E.
      • Stern N.
      Assessment of 5 alpha-reductase activity in hirsute women: comparison of serum androstanediol glucuronide with urinary androsterone and aetiocholanolone excretion.
      ;
      • Joura E.A.
      • Sator M.O.
      • Geusau A.
      • Zeisler H.
      • Söregi G.
      • Huber J.C.
      Die Klinische Wertigkeit von 3 alpha-Androstanediol-Glucuronid bei hilsuten Frauen.
      ). In women, levels of 3α-Adiol glucuronide essentially reflect adrenal precursor levels as well as 5α-reductase activity in peripheral tissues (
      • Vermeulen A.
      • Giagulli V.A.
      Physiology of plasma androstanediol glucuronide.
      ). The correlation between the 3α-Adiol glucuronide and hirsutism score was significant only in hirsute women with increased adrenal androgen secretion (increased DHEA/DHEA-S) and in women with idiopathic hirsutism. The correlation was not significant in hirsute women with increased ovarian testosterone secretion (
      • Pang S.
      • Wang M.
      • Jeffries S.
      • Riddick L.
      • Clark A.
      • Estrada E.
      Normal and elevated 3 alpha-androstanediol glucuronide concentrations in women with various causes of hirsutism and its correlation with degree of hirsutism and androgen levels.
      ). In women with facial hirsutism, serum 3α-Adiol glucuronide concentrations had no correlation with degree of facial hirsutism (
      • Salman K.
      • Spielvoge R.L.
      • Shulman L.H.
      • Miller J.L.
      • Vanderlinde R.E.
      • Rose L.I.
      Serum androstanediol glucuronide in women with facial hirsutism.
      ) and the levels were no more often increased than the other androgen precursors in women with mild to moderate hirsutism (
      • Vermeulen A.
      • Giagulli V.A.
      Physiology of plasma androstanediol glucuronide.
      ;
      • Giagulli V.A.
      • Giorgino R.
      • Vermeulen A.
      Is plasma 5 alpha-androstane 3 alpha, 17 beta-diol glucuronide a biochemical marker of hirsutism in women?.
      ). Modifications in peripheral androgen activity (presumably through 5α-reductase activity) were shown to be time-dependent, and serum 3α-Adiol glucuronide seemed to reflect changes after 6 mo of treatment. (
      • Carmina E.
      • Stanczyk F.Z.
      • Gentzchein E.
      • Lobo R.A.
      Time-dependent changes in serum 3 alpha-androstanediol glucuronide correlate with hirsutism scores after ovarian suppression.
      ). Overall, serum levels of 3α-Adiol glucuronide appears to represent either adrenal androgen production or skin 5 α-reductase activity (
      • Paulson R.J.
      • Serafini P.C.
      • Catalino J.A.
      • Lobo R.A.
      Measurement of 3 alpha, 17 beta-androstanediol glucuronide in serum and urine and the correlation with skin 5 alpha-reductase activity.
      ;
      • Rittmaster R.S.
      • Thompson D.L.
      Effect of leuprolide and dexamethasone on hair growth and hormone levels in hirsute women: the relative importance of the ovary and the adrenal in the pathogenesis of hirsutism.
      ). Although substantial evidence indicated its significance in hirsute women with polycystic ovary syndrome, the routine measurement of serum 3α-Adiol glucuronide is not recommended in the evaluation of idiopathic hirsutism or in other hirsute patients (
      • Azziz R.
      • Carmina E.
      • Sawaya M.E.
      Idiopathic hirsutism.
      ), as this is currently conceptualized to be due to peripherally regionalized in situ hyperandrogenism.
      Androsterone glucuronide/sulfates were also proposed to reflect peripheral androgen metabolism (
      • Matteri R.K.
      • Stanczyk F.Z.
      • Gentzschein E.E.
      • Delgado C.
      • Lobo R.A.
      Androgen sulfate and glucuronide conjugates in nonhirsute and hirsute women with polycystic ovarian syndrome.
      ). In women with idiopathic hirsutism or polycystic ovary disease, androsterone glucuronide was found to be elevated and could reflect an increased production of adrenal androgens, but its level did not correlate with the severity of hirsutism (
      • Thompson D.L.
      • Horton N.
      • Rittmaster R.S.
      Androsterone glucuronide is a marker of adrenal hyperandrogensim in hirsute women.
      ). Androsterone sulfate, being the most abundant 5α-reduced androgen metabolite in serum, however, was not recommended as a marker of either adrenal androgen production or hirsutism (
      • Zwicker H.
      • Rittmaster R.S.
      Androsterone sulfate: physiology and clinical significance in hirsute women.
      ;
      • Azziz R.
      • Carmina E.
      • Sawaya M.E.
      Idiopathic hirsutism.
      ). Table II summarizes the alterations of enzyme activity in androgen-dependent dermatoses.
      Table IIEnzyme activity in androgen-dependent dermatoses
      AcneAndrogenetic alopeciaHirsutism
      Steroid sulfatase↑ (Mo)??
      → (K)
      3β-HSD?↑ (SG, HF)?
      17β-HSD→(SG)↑(HF)?
      5α-reductase→(SG)↑(HF)↑(F)
      Aromatase?↓(HF)?
      3α-HSD???
      ? not known, ↑: increased, ↓: decreased, →: no difference, SG: sebaceous gland; K: keratinocytes, F: fibroblast, Mo: monocytes, HF: hair follicle

      PHARMACOLOGIC DEVELOPMENT OF STEROIDOGENIC ENZYME INHIBITORS

      Steroid sulfatase inhibitors

      As androgens and estrogens may be synthesized inside the cells (or cancer cells) utilizing the circulating systemic precursors DHEA-S and estrone sulfate, therapeutic agents targeted to inhibit steroid sulfatase activity may have therapeutic potential for androgen-sensitive and estrogen-sensitive diseases. Many novel compounds have been developed and evaluated mainly for the treatment of breast cancer (
      • Billich A.
      • Nussbaumer P.
      • Lehr P.
      Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors.
      ;
      • Chetrite G.S.
      • Pasqualini J.R.
      The selective estrogen enzyme modulator (SEEM) in breast cancer.
      ). Steroidal inhibitors may include 17α-substituted benzyl-estradiols, 3-O-sulfamate estrone, 2-methoxyestrone-3-O-sulfamate, the combination of two substituents at positions C3 and C17α of estradiol 3-O-sulfamate, e.g., 17α-benzyl (or 4′-tert-butylbenzyl)estra-1,3,5(10)-trienes and 17α-derivatives of estradiol, 17β-(N-alkylcarbamoyl)-estra-1,3,5(10)-trien-3-O-sulfamates, and 17β-(N-alkanoyl)-estra-1,3,5(10)-trien-3-O-sulfamates (
      • Woo L.W.
      • Howarth N.M.
      • Purohit A.
      • Hejaz H.A.
      • Reed M.J.
      • Potter B.V.
      Steroidal and nonsteroidal sulfamates as potent inhibitors of steroid sulfatase.
      ;
      • Li P.K.
      • Chu G.H.
      • Guo J.P.
      • Peters A.
      • Selcer K.W.
      Development of potent non-estrogenic estrone sulfatase inhibitors.
      ;
      • Ciobanu L.C.
      • Boivin R.P.
      • Luu-The V.
      • Labrie F.
      • Poirier D.
      Potent inhibition of steroid sulfatase activity by 3-O-sulfamate 17 alpha-benzyl(or 4′-tert-butylbenzyl)estra-1,3,5(10)-trienes: combination of two substituents at positions C3 and C17alpha of estradiol.
      ;
      • Boivin R.P.
      • Luu-The V.
      • Lachance R.
      • Labrie F.
      • Poirier D.
      Structure–activity relationships of 17alpha-derivatives of estradiol as inhibitors of steroid sulfatase.
      ;
      • Purohit A.
      • Woo L.W.
      • Barrow D.
      • Hejaz H.A.
      • Nicholson R.I.
      • Potter B.V.
      • Reed M.J.
      Non-steroidal and steroidal sulfamates: new drugs for cancer therapy.
      ). Nonsteroidal sulfamates such as ( p-O-sulfamoyl)-N-alkanoyl-tyramines, tricyclic coumarin sulfamates, tricyclic oxepin sulfamate, and substituted chromenone sulfamates (e.g., sulfamic acid 2-t-butyl-4-oxo-4H-chromen-6-yl ester) have been described (
      • Billich A.
      • Nussbaumer P.
      • Lehr P.
      Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors.
      ;
      • Purohit A.
      • Woo L.W.
      • Barrow D.
      • Hejaz H.A.
      • Nicholson R.I.
      • Potter B.V.
      • Reed M.J.
      Non-steroidal and steroidal sulfamates: new drugs for cancer therapy.
      ).
      Estrone-3-O-sulfamate was shown to inhibit the steroid sulfatase enzyme activity in a concentration-dependent manner in human hair DP ex vivo (
      • Hoffmann R.
      • Rot A.
      • Niiyama S.
      • Billich A.
      Steroid sulfatase in the human hair follicle concentrations in the dermal papilla.
      ).

      3β-HSD inhibitors

      Known inhibitors used clinically in dermatology include the gestagens cyproterone acetate, norgestrel, norethisterone, which exhibit a dual activity by parallel binding to a androgen receptor (
      • Dumont M.
      • Luu-The V.
      • Dupont E.
      • Pelletier G.
      • Labrie F.
      Characterization, expression, and immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in human skin.
      ;
      • Zouboulis ChC
      • Seltmann H.
      • Neitzel H.
      • Orfanos C.E.
      Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).
      ). Trilostane (4α-5-epoxy-17β-hydroxy-3-oxo-5α-androstan-2-carbonitrile) and cyanoketone (2α-cyano-17β-hydroxy-4,4,17α-trimethylandrost-5-en-3-one) are two classical steroidal inhibitors of type 1 3β-HSD (
      • Cooke G.M.
      Differential effects of trilostane and cyanoketone on the 3 beta-hydroxysteroid dehydrogenase-isomerase reactions in androgen and 16-androstene biosynthetic pathways in the pig testis.
      ). Trilostane can attenuate the preovulatory gonadotropin surge by inhibiting progesterone synthesis (
      • Mahesh V.B.
      • Brann D.W.
      Regulation of the preovulatory gonadotropin surge by endogenous steroids.
      ). It has also been clinically tried to treat Cushing's syndrome (
      • Engelhardt D.
      • Weber M.M.
      Therapy of Cushing's syndrome with steroid biosynthesis inhibitors.
      ). Isoflavonoids such as genistein and daidzein were also shown to exert an anti-3β-HSD effect (
      • Le Bail J.C.
      • Champavier Y.
      • Chulia A.J.
      • Habrioux G.
      Effects of phytoestrogens on aromatase, 3beta and 17beta-hydroxysteroid dehydrogenase activities and human breast cancer cells.
      ). Thiazolidinediones was recently shown to inhibit directly the steroidogenic enzymes P450c17 and type 2 3β-HSD (
      • Arlt W.
      • Auchus R.J.
      • Miller W.L.
      Thiazolidinediones but not metformin directly inhibit the steroidogenic enzymes P450c17 and 3beta-hydroxysteroid dehydrogenase.
      ).

      17β-HSD inhibitors

      As the 17β-HSD system plays a key part in the formation or inactivation of several active androgens and estrogens from circulating precursors, these isoenzymes can regulate tumor cell proliferation in androgen- and estrogen-dependent cancers. Endocrine therapies for the treatment and prevention of breast cancer are presently under intensive clinical trials with the intention of developing dual-action compounds to block estrogen action optimally by antagonizing the estrogen receptor as well as inhibiting the estradiol biosynthesis (
      • Blomquist C.H.
      Kinetic analysis of enzymic activities: prediction of multiple forms of 17 beta-hydroxysteroid dehydrogenase.
      ;
      • Tremblay M.R.
      • Poirier D.
      Overview of a rational approach to design type I 17beta-hydroxysteroid dehydrogenase inhibitors without estrogenic activity: chemical synthesis and biological evaluation.
      ).
      As stated before, various 17β-HSD isoenzymes possess different catalytic reductive or oxidative activity, thus design of specific antagonist should aim at binding to hydrophilic and cofactor-depending sites at the active center of the isoenzyme (
      • Krazeisen A.
      • Breitling R.
      • Moller G.
      • Adamski J.
      Phytoestrogens inhibit human 17 beta-hydroxysteroid dehydrogenase type 5.
      ). Prior to better characterization of the molecular genetics and physiology of different isoenzymes, the exploration and development of specific enzyme inhibitors were performed depending on the selected tissues (liver, ovary, placenta, testis or prostate, etc.) from different species under examination (human vs rat, mouse or porcine). Specificity of the compounds based on the analysis of these data seems to be confounded by the finding that the same organ/tissue may contain more than one 17β-HSD isoenzyme (
      • Luu-The V.
      Analysis and characteristics of multiple types of human 17β-hydroxysteroid dehydrogenase.
      ).
      Well characterized 17β-HSD1 inhibitors include estradiol derivatives containing a bromopropyl/or iodopropyl group at position 16α such as 16-(bromoalkyl)-estradiols (
      • Luu-The V.
      • Zhang Y.
      • Poirier D.
      • Labrie F.
      Characteristics of human types 1, 2 and 3 17 beta-hydroxysteroid dehydrogenase activities: oxidation/reduction and inhibition.
      ;
      • Tremblay M.R.
      • Auger S.
      • Poirier D.
      Synthesis of 16-(bromoalkyl)-estradiols having inhibitory effect on human placental estradiol 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD type 1).
      ), phytoestrogens such as flavonoids (including flavone, flavanone, and isoflavone), isoflavonoids, and lignans (
      • Evans B.A.
      • Griffiths K.
      • Morton M.S.
      Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids.
      ;
      • Makela S.
      • Poutanen M.
      • Lehtimaki J.
      • Kostian M.L.
      • Santti R.
      • Vihko R.
      Estrogen-specific 17 beta-hydroxysteroid oxidoreductase type 1 (E.C. 1.1.1.62) as a possible target for the action of phytoestrogens.
      ,
      • Makela S.
      • Poutanen M.
      • Kostian M.L.
      • Lehtimaki N.
      • Strauss L.
      • Santti R.
      • Vihko R.
      Inhibition of 17beta-hydroxysteroid oxidoreductase by flavonoids in breast and prostate cancer cells.
      ;
      • Le Bail J.C.
      • Laroche T.
      • Marre-Fournier F.
      • Habrioux G.
      Aromatase and 17beta-hydroxysteroid dehydrogenase inhibition by flavonoids.
      ), trifluoromethylacetylenic secoestradiol (
      • Lawate S.S.
      • Covey D.F.
      Trifluoromethylacetylenic alcohols as affinity labels: inactivation of estradiol dehydrogenase by a trifluoromethylacetylenic secoestradiol.
      ), and 6β-(thiaheptanamide) derivatives of estradiol (
      • Poirier D.
      • Dionne P.
      • Auger S.
      A 6beta-(thiaheptanamide) derivative of estradiol as inhibitor of 17beta-hydroxysteroid dehydrogenase type 1.
      ). Compounds belonging to this group might also include those described to inactivate ovarian 17β-HSD isoenzyme, such as glycyrrhizin and glycyrrhetinic acid (
      • Sakamoto K.
      • Wakabayashi K.
      Inhibitory effect of glycyrrhetinic acid on testosterone production in rat gonads.
      ;
      • Armanini D.
      • Bonanni G.
      • Palermo M.
      Reduction of serum testosterone in men by licorice.
      ) or to inhibit the enzyme activity in breast cancer cell lines, such as medrogestone (
      • Chetrite G.S.
      • Ebert C.
      • Wright F.
      • Philippe J.C.
      • Pasqualini J.R.
      Effect of Medrogestone on 17beta-hydroxysteroid dehydrogenase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines.
      ).
      Well characterized 17β-HSD2 inhibitors containing a spiro-gs-lactone at position 17 (
      • Luu-The V.
      • Zhang Y.
      • Poirier D.
      • Labrie F.
      Characteristics of human types 1, 2 and 3 17 beta-hydroxysteroid dehydrogenase activities: oxidation/reduction and inhibition.
      ), 7α-thioalkyl and 7α-thioaryl derivatives of spironolactone such as 3-Oxo-17α-pregna-4-ene-7α-{4-[2-(1-pipendiryl)ethoxy]-benzylthio} 21,17-carbolactone (
      • Tremblay M.R.
      • Luu-The V.
      • Leblanc G.
      • Noel P.
      • Breton E.
      • Labrie F.
      • Poirier D.
      Spironolactone-related inhibitors of type II 17beta-hydroxysteroid dehydrogenase: chemical synthesis, receptor binding affinities, and proliferative/antiproliferative activities.
      ), N-butyl-N-methyl-11-(3′-hydroxy-21′, 17′-carbolac-tone-19′-nor-17′α-pregna-1′,3′,5′(10′)-trien-7′α-yl)-undecanamide (
      • Sam K.M.
      • Labrie F.
      • Poirier D.
      N-Butyl-N-methyl-11-(3′-hydroxy-21′,17′-carbolactone-19′-nor-17′alpha-pregna-1′,3′,5′(10′)-trien-7′alpha-yl)-undecanamide: an inhibitor of type 2 17beta-hydroxysteroid dehydrogenase that does not have oestrogenic or androgenic activity.
      ), flavonoids, isoflavonoids, and lignans (
      • Evans B.A.
      • Griffiths K.
      • Morton M.S.
      Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids.
      ;
      • Makela S.
      • Poutanen M.
      • Kostian M.L.
      • Lehtimaki N.
      • Strauss L.
      • Santti R.
      • Vihko R.
      Inhibition of 17beta-hydroxysteroid oxidoreductase by flavonoids in breast and prostate cancer cells.
      ;
      • Le Bail J.C.
      • Laroche T.
      • Marre-Fournier F.
      • Habrioux G.
      Aromatase and 17beta-hydroxysteroid dehydrogenase inhibition by flavonoids.
      ). Based on the characterized tissue distribution of the isoenzymes, included in this group might be compounds that are able to work on:
      1 The placenta: danazol; ethinylestradiol (
      • Blomquist C.H.
      • Lindemann N.J.
      • Hakanson E.Y.
      Inhibition of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activities of human placenta by steroids and non-steroidal hormone agonists and antagonists.
      ); unsaturated fatty acids, such as oleic, arachidonic, linoleic, and linolenic acid (
      • Blomquist C.H.
      • Lindemann N.J.
      • Hakanson E.Y.
      Inactivation of soluble 17 beta-hydroxysteroid dehydrogenase of human placenta by fatty acids.
      ); periodate-oxidized NADP+ (
      • Mendoza-Hernandez G.
      • Lopez-Solache I.
      • Diaz-Zagoya J.C.
      Periodate-oxidized NADP+ is a powerful inhibitor of human placental estradiol-17 beta dehydrogenase.
      ); 14,15-secoestra-1,3,5(10)-trien-15-ynes (
      • Auchus R.J.
      • Palmer J.O.
      • Carrell H.L.
      • Covey D.F.
      Preparation of 14,15-secoestra-1,3,5(10)-trien-15-ynes, inhibitors of estradiol dehydrogenase.
      ); spiro-gp-lactones containing the C-18 nucleus (
      • Sam K.M.
      • Auger S.
      • Luu-The V.
      • Poirier D.
      Steroidal spiro-gamma-lactones that inhibit 17 beta-hydroxysteroid dehydrogenase activity in human placental microsomes.
      ); and chalcones, such as naringenin chalcone and 4-hydroxychalcone (
      • Le Bail J.C.
      • Pouget C.
      • Fagnere C.
      • Basly J.P.
      • Chulia A.J.
      • Habrioux G.
      Calcones are potent inhibitors of aromatase and 17beta-hydroxysteroid dehydrogenase activities.
      ).
      2 Human benign prostatic hyperplasia tissue: testolactone (
      • Bartsch W.
      • Klein H.
      • Sturenburg H.J.
      • Voigt K.D.
      Metabolism of androgens in human benign prostatic hyperplasia: aromatase and its inhibition.
      ).
      3 Liver microsomal enzymes: nonsteroidal anti-inflammatory agents and nonsteroidal estrogens, such as hexestrol, dienstrol, diethylstilbestrol, and zearalenone (
      • Hasebe K.
      • Hara A.
      • Nakayama T.
      • Hayashibara M.
      • Inoue Y.
      • Sawada H.
      Inhibition of hepatic 17 beta- and 3 alpha-hydroxysteroid dehydrogenases by antiinflammatory drugs and nonsteroidal estrogens.
      ); and retinoids (13-cis retinoic acid >9-cis retinoic acid > all-trans retinoic acid) (
      • Murray M.
      • Butler A.M.
      • Martini R.
      Inhibition of microsomal 17 beta-hydroxysteroid oxidoreduction activities in rat liver by all-trans-, 9-cis- and 13-cis-retinoic acid.
      ).
      Well-characterized 17β-HSD3 inhibitors include 1,4-androstadiene-1,6,17-trione (
      • Luu-The V.
      • Zhang Y.
      • Poirier D.
      • Labrie F.
      Characteristics of human types 1, 2 and 3 17 beta-hydroxysteroid dehydrogenase activities: oxidation/reduction and inhibition.
      ) and androsterone 3β-substituted derivatives (
      • Ngatcha B.T.
      • Luu-The V.
      • Poirier D.
      Androsterone 3beta-substituted derivatives as inhibitors of type 3 17beta-hydroxysteroid dehydrogenase.
      ). Included might also be those reported to inhibit testicular 17β-HSD enzyme activity, such as licorice (glycyrrhizin and glycyrrhetinic acid) (
      • Sakamoto K.
      • Wakabayashi K.
      Inhibitory effect of glycyrrhetinic acid on testosterone production in rat gonads.
      ;
      • Armanini D.
      • Bonanni G.
      • Palermo M.
      Reduction of serum testosterone in men by licorice.
      ), losulazine (
      • Ray A.
      • Chatterjee S.
      • Biswas N.M.
      Study on the activities of testes and accessory sex glands after losulazine treatment in rats.
      ), amphetamine (
      • Tsai S.C.
      • Chen J.J.
      • Chiao Y.C.
      • et al.
      The role of cyclic AMP production, calcium channel activation and enzyme activities in the inhibition of testosterone secretion by amphetamine.
      ), methotrexate (
      • Badri S.N.
      • Vanithakumari G.
      • Malini T.
      Studies on methotrexate effects on testicular steroidogenesis in rats.
      ), and S-petasine (like a sesquiterpene ester, being an anti-inflammatory analgesic component of the butterbur, Petasites hybridus;
      • Lin H.
      • Chien C.H.
      • Lin Y.L.
      • Chen C.F.
      • Wang P.S.
      Inhibition of testosterone secretion by S-petasin in rat testicular interstitial cells.
      ).

      5α-reductase inhibitors

      Development of specific 5α-reductase inhibitors began soon after 5α-DHT was reported to be the major androgen acting in the periphery (
      • Liang T.
      • Rasmusson G.H.
      • Brooks J.R.
      Biochemical and biological studies with 4-azasteroidal 5α-reductase inhibitors.
      ,
      • Liang T.
      • Heiss C.E.
      • Cheung A.H.
      • Reynolds G.F.
      • Rasmusson G.H.
      4-azasteroidal 5 alpha-reductase inhibitors without affinity for the androgen receptor.
      ). Great progress has since been made and continuing interests grow in developing more potent and specific 5α-reductase inhibitors. As 5α-DHT, being two to 10 times stronger than testosterone in androgenicity, was supposed to play a more important part than testosterone in many androgen-dependent diseases (benign prostata hyperplasia, prostatic carcinoma, acne, androgenetic alopecia, hirsutism;
      • Chen W.
      • Zouboulis C.C.
      • Orfanos C.E.
      The 5 alpha-reductase system and its inhibitors. Recent development and its perspective in treating androgen-dependent skin disorders.
      ;
      • Bartsch G.
      • Rittmaster R.S.
      • Klocker H.
      Dihydrotestosterone and the concept of 5 alpha-reductase inhibition in human benign prostatic hyperplasia.
      ), whereas specific inhibition of its formation would bring the advantage of sparing the anti-virilizing side-effects of general androgen receptor blockers (e.g., flutamide). Inhibitors can be classified based on the chemical structures of steroidal vs nonsteroidal inhibitors or according to the isoenzyme specificity as type 1, type 2 and type-1/2 dual inhibitors (
      • Chen W.
      • Zouboulis C.C.
      • Orfanos C.E.
      The 5 alpha-reductase system and its inhibitors. Recent development and its perspective in treating androgen-dependent skin disorders.
      ;
      • Zouboulis ChC
      Antiandrogene Therapie-Neue Entwicklungen der systemischen und peripheren Inhibition.
      ). It was noticed that the Ki values vary depending on the species examined (human vs rat) and the cell/tissue origin tested (e.g., testis vs prostate or primary culture vs cell lines). Except for finasteride, most of these synthetic chemicals or phytotherapeutic agents are still undergoing in vitro tests, animal studies, or clinical trials (
      • Zouboulis ChC
      Antiandrogene Therapie-Neue Entwicklungen der systemischen und peripheren Inhibition.
      ). Finasteride, a specific type 2 5α-reductase competitive inhibitor, is the first systemic drug approved for clinical use and has been shown to be effective for the treatment of androgenetic alopecia in young to middle-aged men as well as aged men between 53 and 76 y, but not in post-menopausal women (
      • Brenner S.
      • Matz H.
      Improvement in androgenetic alopecia in 53–76-year old men using oral finasteride.
      ;
      • Whiting D.A.
      • Waldstreicher J.
      • Sanchez M.
      • Kaufman K.D.
      Measuring reversal of hair miniaturization in androgenetic alopecia by follicular counts in horizontal sections of serial scalp biopsies: results of finasteride 1 mg treatment of men and postmenopausal women.
      ).
      Specific type 1 inhibitors include certain steroidal inhibitors, such as 4-azasteroids (e.g., MM-386: 4,7β-dimethyl-4-aza-5α-cholestan-3-one) or 6-azasteroidal 17β-carboxamide triaryls (
      • Li X.
      • Chen C.
      • Singh S.M.
      • Labrie F.
      The enzyme and inhibitors of 4-ene-3-oxosteroid 5α-oxidoreductase.
      ), and nonsteroidal inhibitors benzoquinolinones (LY 191704: 8-chloro-4-methyl-1,2,3,4,4a,5,6,10b-octaahydro-benzo[f]quinolin-3(2H)-one), 6-[4-(N,N-diisopropylcarbamoyl) phenyl]-N-methyl-quinolin-2-one 5) (
      • Baston E.
      • Palusczak A.
      • Hartmann R.W.
      6-substituted 1H-quinolin-2-ones and 2-methoxy-quinolines: synthesis and evaluation as inhibitors of steroid 5 alpha-reductases types 1 and 2.
      ), benzo [c]quinolizin-3-ones (
      • Guarna A.
      • Machetti F.
      • Occhiato E.G.
      • et al.
      Benzo[c]quinolizin-3-ones: a novel class of potent and selective nonsteroidal inhibitors of human steroid 5alpha-reductase 1.
      ), certain plant extracts, such as green tea extract catechins (epicatechin-3-gallate and epigallocatechin-3-gallate) (
      • Liao S.
      • Hiipakka R.A.
      Selective inhibition of steroid 5α-reductase isozymes by tea epicatechin-3-gallate and epigallocatechin-3-gallate.
      ), and suramin, zinc, and azelaic acid (
      • Chen W.
      • Zouboulis C.C.
      • Orfanos C.E.
      The 5 alpha-reductase system and its inhibitors. Recent development and its perspective in treating androgen-dependent skin disorders.
      ). MK-386, an azasteroid that specifically inhibits the type 1 5α-reductase in vitro, was shown to suppress sebum as well as serum 5α-DHT in a concentration-dependent manner (
      • Schwartz J.I.
      • Tanaka W.K.
      • Wang D.Z.
      • et al.
      MK-386, an inhibitor of 5 alpha-reductase type 1, reduces dihydrotestosterone concentrations in serum and sebum without affecting dihydrotestosterone concentrations in semen.
      ;
      • Baston E.
      • Palusczak A.
      • Hartmann R.W.
      6-substituted 1H-quinolin-2-ones and 2-methoxy-quinolines: synthesis and evaluation as inhibitors of steroid 5 alpha-reductases types 1 and 2.
      ).
      Specific type 2 inhibitors, in addition to finasteride, include 4-methyl-4-azasteroids (e.g., turosteride: 1-(4-methyl-3-oxo-4-aza-5α-androstane-17β-carbonyl)-1,3-diisopropylurea, or MK-963: [5α-23-methyl-4-aza-21-norchol-1-ene-3,20-dione])
      Seiffert K, Fritsch M, Zoubouls ChC: 5α-reductase inhibitors exhibit distinct effects on human keratinocytes and sebocytes in vitro. J Invest Dermatol 110:550, 1998 (Abstr.)
      (
      • Chen W.
      • Zouboulis C.C.
      • Orfanos C.E.
      The 5 alpha-reductase system and its inhibitors. Recent development and its perspective in treating androgen-dependent skin disorders.
      ), 4-azasteroids (e.g., MK-434: 17β-benzoyl-4-aza-5α-androst-1-en-3-one, dihydrofinasteride)
      Seiffert K, Fritsch M, Zoubouls ChC: 5α-reductase inhibitors exhibit distinct effects on human keratinocytes and sebocytes in vitro. J Invest Dermatol 110:550, 1998 (Abstr.)
      (
      • Chen W.
      • Zouboulis C.C.
      • Orfanos C.E.
      The 5 alpha-reductase system and its inhibitors. Recent development and its perspective in treating androgen-dependent skin disorders.
      ;
      • Zouboulis ChC
      Antiandrogene Therapie-Neue Entwicklungen der systemischen und peripheren Inhibition.
      ), chlormadinone acetate (
      • Nukui F.
      Effects of chlormadinone acetate and ethinylestradiol treatment on epididymal 5 alpha-reductase activities in patients with prostate cancer.
      ), osaterone acetate (TZP-4238: 17α-acetoxy-chloro-2-oxa-4,6-pregnadiene-3,20-dione) (
      • Takezawa Y.
      • Fukabori Y.
      • Yamanaka H.
      • Mieda M.
      • Honma S.
      • Kushitani M.
      • Hamataki N.
      Effects of the new steroidal antiandrogen TZP-4238 on hormone-induced canine prostatic hyperplasia.
      ), epristeride (SK&F 105657 or ONO-9302: N-(t-butyl)androst-3,5-diene-17β-carboxamide-3-carboxylic acid,) (
      • Levy M.A.
      • Brandt M.
      • Sheedy K.M.
      • et al.
      Epristeride is a selective and specific uncompetitive inhibitor of human steroid 5 alpha-reductase isoform 2.
      ), 17α-estradiol (
      • Hevert F.
      17α-Estradiol-ein moderner Inhibitor der 5α-reductase.
      ), 6-Methylenesteroidal derivatives (
      • Li X.
      • Chen C.
      • Singh S.M.
      • Labrie F.
      The enzyme and inhibitors of 4-ene-3-oxosteroid 5α-oxidoreductase.
      ), 17-(5′-isoxazolyl)androsta-4,16-dien-3-one (L-39) (
      • Nnane I.P.
      • Long B.J.
      • Ling Y.Z.
      • Grigoryev D.N.
      • Brodie A.M.
      Anti-tumor effects and pharmacokinetic profile of 17-(5′-isoxazolyl)androsta-4,16-dien-3-one (L-39) in mice: an inhibitor of androgen synthesis.
      ), nonsteroidal compounds such as 6-[4-(N,N-diisopro-pylcarbamoyl)phenyl]-1H-quinolin-2-one 4 (
      • Baston E.
      • Palusczak A.
      • Hartmann R.W.
      6-substituted 1H-quinolin-2-ones and 2-methoxy-quinolines: synthesis and evaluation as inhibitors of steroid 5 alpha-reductases types 1 and 2.
      ), and tricyclic compounds such as 4-[3-[5-benzyl-8-(2-methyl)propyl-10,11-dihydrodibenz[b,f ]azepine-2-carboxamido] phenoxy]butyric acid (
      • Takami H.
      • Nonaka H.
      • Kishibayashi N.
      • Ishii A.
      • Kase H.
      • Kumazawa T.
      Synthesis of tricyclic compounds as steroid 5 alpha-reductase inhibitors.
      ), etc.
      Several novel compounds with potent dual inhibitory activity on both type 1 and type 2 isoenzymes have been reported. Steroidal antagonists include N-(1,1,1,3,3,3-hexafluorophenyl-pro-pyl)-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide (PNU 157706) (
      • di Salle E.
      • Giudici D.
      • Radice A.
      • et al.
      PNU 157706, a novel dual type I and type II 5α-reductase inhibitor.
      ) dutasteride (
      • Frye S.V.
      • Bramson H.N.
      • Hermann D.J.
      • Lee F.W.
      • Sinhababu A.K.
      • Tian G.
      Discovery and development of GG745, a potent inhibitor of both isozymes of 5 alpha-reductase.
      ;
      • Gisleskog P.O.
      • Hermann D.
      • Hammarlund-Udenaes M.
      • Karlsson M.O.
      A model for the turnover of dihydrotestosterone in the presence of the irreversible 5 alpha-reductase inhibitors GI198745 and finasteride.
      ), oxendolone (TSAA-291: 16β-ethyl-17β-hydroxy-4-estren-3-one) (
      • Sudo K.
      • Yoshida K.
      • Akinaga Y.
      • Nakayama R.
      5alpha-reduction of an anti-androgen TSAA-291, 16beta-ethyl-17beta-hydroxy-4-estren-3-one, by nuclear 5alpha-reductase in rat prostates.
      ;
      • Li X.
      • Chen C.
      • Singh S.M.
      • Labrie F.
      The enzyme and inhibitors of 4-ene-3-oxosteroid 5α-oxidoreductase.
      ), 19-nor-10-azasteroids (
      • Guarna A.
      • Belle C.
      • Machetti F.
      • et al.
      19-nor-10-azasteroids: a novel class of inhibitors for human steroid 5alpha-reductases 1 and 2.
      ), and progesterone-based steroids bearing an oxime group connected to the steroidal D-ring (
      • Hartmann R.W.
      • Hector M.
      • Haidar S.
      • Ehmer P.B.
      • Reichert W.
      • Jose J.
      Synthesis and evaluation of novel steroidal oxime inhibitors of P450 17 (17 alpha-hydroxylase/C17-20-lyase) and 5 alpha-reductase types 1 and 2.
      ). Nonsteroidal compounds include benzoquinolinone (e.g., LY320236) (
      • McNulty A.M.
      • Audia J.E.
      • Bemis K.G.
      • Goode R.L.
      • Rocco V.P.
      • Neubauer B.L.
      Kinetic analysis of LY320236: competitive inhibitor of type I and non-competitive inhibitor of type II human steroid 5 alpha-reductase.
      ), plant extracts such as Serenoa repens extract permixon (
      • Bayne C.W.
      • Ross M.
      • Donnelly F.
      • Habib F.K.
      The selectivity and specificity of the actions of the lipido-sterolic extract of Serenoa repens (Permixon) on the prostate.
      ), Artocarpus incisus (
      • Shimizu K.
      • Fukuda M.
      • Kondo R.
      • Sakai K.
      The 5 alpha-reductase inhibitory components from heartwood of Artocarpus incisus: structure–activity investigations.
      ), isoflavonoids and lignans (
      • Evans B.A.
      • Griffiths K.
      • Morton M.S.
      Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids.
      ), alizarin and curcumin (
      • Liao S.
      • Lin J.
      • Dang M.T.
      • Zhang H.
      • Kao Y.-H.
      • Fukuchi J.
      • Hiipakka R.A.
      Growth suppression of hamster flank organs by topical application of catechins, alizarin, curcumin, and myristoleic acid.
      ), phenazine derivatives (e.g., riboflavin) (
      • Li X.
      • Chen C.
      • Singh S.M.
      • Labrie F.
      The enzyme and inhibitors of 4-ene-3-oxosteroid 5α-oxidoreductase.
      ), myristoleic acid (
      • Liao S.
      • Lin J.
      • Dang M.T.
      • Zhang H.
      • Kao Y.-H.
      • Fukuchi J.
      • Hiipakka R.A.
      Growth suppression of hamster flank organs by topical application of catechins, alizarin, curcumin, and myristoleic acid.
      ) and γ-linolenic acid (
      • Liang T.
      • Liao S.
      Inhibition of steroid 5α-reductase by specific aliphatic unsaturated fatty acids.
      ,
      • Liang T.
      • Liao S.
      Growth suppression of hamster flank organs by topical application of γ-linolenic acid and other fatty acid inhibitors of 5α-reductase.
      ;
      • Liao S.
      • Hiipakka R.A.
      Selective inhibition of steroid 5α-reductase isozymes by tea epicatechin-3-gallate and epigallocatechin-3-gallate.
      ), indole derivatives such as 4-[3-[3-[bis(4-isobutylphenyl)-methylamino]benzoyl]-1H-indol-1-yl] butyric acid (FK 143) (
      • Katashima M.
      • Irino T.
      • Shimojo F.
      • et al.
      Pharmacokinetics and pharmacodynamics of FK143, a nonsteroidal inhibitor of steroid 5 alpha-reductase, in healthy volunteers.
      ), sodium-4-[2-(2,3-dimethyl-4-and 2 over black square); [1 and 2 over black square]-(4- isobutylphenyl)ethoxy]benzolamino]phenoxy] butyrate (ONO-3805) (
      • Takahashi O.
      • Imai K.
      • Watanabe K.
      • et al.
      [The effect of sodium-4-[2-(2,3-dimethyl-4-[1-(4-isobutylphenyl)ethoxy]benzolamino)phenoxy] butyrate (ONO-3805) and antiandrogenic agents on the rat accessory sex organs].
      ), indoline and aniline derivatives (
      • Igarashi S.
      • Inami H.
      • Hara H.
      • Koutoku H.
      • Oritani H.
      • Mase T.
      A novel class of inhibitors for human and rat steroid 5alpha-reductases: synthesis and biological evaluation of indoline and aniline derivatives. III.
      ). γ-Linolenic acid was shown to inhibit testosterone-stimulated flank organ growth but not 5α-DHT-stimulated flank organ growth (
      • Liang T.
      • Liao S.
      Growth suppression of hamster flank organs by topical application of γ-linolenic acid and other fatty acid inhibitors of 5α-reductase.
      ). CS-891 inhibits 5α-reductase activity in freshly isolated DP of human hair follicles (
      • Niiyama S.
      • Kojima K.
      • Hamada T.
      • Happle R.
      • Hoffmann R.
      The novel drug CS-891 inhibits 5 alpha-reductase activity in freshly isolated dermal papilla of human hair follicles.
      ). It remains to be determined if these dual inhibitors can further decrease the tissue 5α-DHT level and are clinically more effective than finasteride in the treatment of male androgenetic alopecia.
      Table III and Figure 2 show the representative specific steroidogenic enzyme inhibitors and their chemical structures, respectively.
      Table IIISpecific inhibitors of steroidogenic isoenzymes
      Steroidogenic enzymesIsoformsCharacterized inhibitors
      SteroidalNon-steroidal
      Steroid sulfatase17α-substituted benzylestradiolstricyclic coumarin sulfamates
      Estrone-3-O-sulfamatetricyclic oxepin sulfamate
      2-methoxyestrone-3-O-sulfamatesubstituted chromenone sulfamates
      17α-benzyl (or 4′-tert-butylbenzyl)estra-1,3,5(10)-trienes
      17β-(N-alkylcarbamoyl)-estra-1,3,5(10)-trien-3-O-sulfamates
      3β-hydroxysteroid dehydrogenaseType 1trilostanecyanoketone
      cyproterone acetateisoflavonoids (genistein)
      norgestrel
      norethindrone
      Type 2thiazolidinediones
      17β-hydroxysteroid dehydrogenaseType 116-(bromoalkyl)-estradiols
      flavonoids, isoflavonoids, lignans
      trifluoromethylacetylenic secoestradiol
      6β-(thiaheptanamide) derivatives of estradiol
      Type 2estrone containing a spiro-gamma-lactone at position 17
      7α-thioalkyl and 7α-thioaryl derivatives of spironolactone
      N-butyl-N-methyl-11-(3′-hydroxy-21′, 17′-carbolactone-19′-nor-17′α-pregna-1′,3′, 5′(10′)-trien-7′α-yl)-undecanamide
      flavonoids, isoflavonoids, lignans
      Type 31,4-androstadiene-1,6,17- trione
      androsterone 3β-substituted derivatives
      5α-reductaseType 14-azasteroids (MK386) 6-azasteroidal 17β-carboxamide triaryls)8-chloro-4-methyl-1,2,3,4,4a,5,6,10b-octaahydro-benzo[f]quinolin-3(2 H)-one (LY 191704)
      6-[4-(N,N-diisopropylcarbamoyl)phenyl]-N-methyl-quinolin-2-one 5)
      benzo[c]quinolizin-3-ones
      epicatechin-3-gallate, epigallocatechin-3-gallate
      suramin
      zinc
      azelaic acid
      Type 2finasteride6-[4-(N,N-diisopropylcarbamoyl)phenyl]-1H-quinolin-2-one 4
      turosteride4-[3-[5-benzyl-8-(2-methyl)propyl-10,11-dihydrodibenz[b,f]azepine-2-carboxamido]phenoxy]butyric acid
      MK-434
      MK-963
      dihydrofinasteride
      chlormadinone acetate
      TZP-4238
      epristeride (SK&F 105657, ONO-9302)
      17α-estradiol
      17-(5′-isoxazolyl)androsta-4,16-dien-3-one
      Type 1/2N-(1,1,1,3,3,3-hexafluorophenyl-propyl)-3-oxo-4-aza- 5α-androst-1-ene-17β-carboxamide (PNU 157706)benzoquinolinone
      dutasterideSerenoa repens extract permixon
      oxendolone (TSAA-291: 16 β-ethyl-17β-hydroxy-4-estren-3-one)Artocarpus incisus
      19-nor-10-azasteroidsisoflavonoids and lignans
      progesterone-based steroids bearing an oxime group connected to the steroidal D-ringalizarin and curcumin
      phenazine derivatives
      myristoleic acid
      γ-linolenic acid
      4-[3-[3-[bis(4-isobutylphenyl)-methylamino]benzoyl]-1H-indol-1-yl] butyric acid (FK 143)
      Figure thumbnail gr2
      Figure 2Chemical structures of some representative steroidogenic enzyme inhibitors.
      Figure thumbnail gr3
      Figure 2Chemical structures of some representative steroidogenic enzyme inhibitors.

      CONCLUSIONS

      Similar to the classical steroidogenic organs, such as gonads and adrenal glands, the skin and its appendages, including hair follicles, sebaceous glands, and eccrine/apocrine glands, are armed with all the necessary enzymes required for androgen synthesis and metabolism. Steroid sulfatase, 3β-HSD1, 17β-HSD3, and the type 1 5α-reductase are the major steroidogenic enzymes responsible for the formation of potent androgens, whereas 17β-HSD2, 3α-HSD, and aromatase seem to inactivate the excess androgens locally in order to achieve androgen homeostasis (
      • Fritsch M.
      • Orfanos C.E.
      • Zouboulis ChC
      Sebocytes are the key regulators of androgen homeostasis in human skin.
      ). Small levels of some isoenzymes found in normal states, may have important implications in disease states, where these factors may be upregulated, contributing to the exaggeration of “peripheral hyperandrogensim”. Clarification of the cutaneous expression and tissue-specific regulation of these steroidogenic isoenzymes could help us to understand better their roles in the growth and development of the skin as well as the pathophysiology of androgen-dependent dermatoses. Finasteride is the first specific isoenzyme inhibitor used for clinical intervention. Rapid pharmacologic advancement in this field and design for other specific potent antagonists foresees a better control or even chemoprevention of acne, hirsutism, and androgenetic alopecia in the near future.

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