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Cytochrome b₅ Expression in Gonadectomy-induced Adrenocortical Neoplasms of the Domestic Ferret (Mustela putorius furo)

Departments of Pediatrics and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO (SW, MH, DBW), University of Applied Sciences Mannheim, Mannheim, Germany (SW), GlaxoSmithKline, Research Triangle Park, NC (RAP), Department of Pathobiology and Diagnostic Investigation, Michigan State University College of Veterinary Medicine, Lansing, MI (MK), Children's Hospital, Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland (MH)

Abstract

Whereas the adrenal glands of healthy ferrets produce only limited amounts of androgenic steroids, adrenocortical neoplasms that arise in neutered ferrets typically secrete androgens or their derivative, estrogen. The 17,20-lyase activity of cytochrome P450 17-hydroxylase/17,20-lyase (P450c17) must increase to permit androgen biosynthesis in neoplastic adrenal tissue. We screened ferret adrenocortical tumor specimens for expression of cytochrome b₅ (cyt b₅), an allosteric regulator that selectively enhances the 17,20-lyase activity of P450c17. Cyt b₅ immunoreactivity was evident in 24 of 25 (96%) adrenocortical adenomas/carcinomas from ferrets with signs of ectopic sex steroid production. Normal adrenocortical cells lacked cyt b₅, which may account for the low production of adrenal androgens in healthy ferrets. Other markers characteristic of gonadal somatic cells, such as luteinizing hormone receptor, aromatase, and GATA4, were coexpressed with cyt b₅ in some of the tumors. We concluded that cyt b₅ is upregulated during gonadectomy-induced adrenocortical neoplasia and is a marker of androgen synthetic potential in these tumors.

Key words: Adrenal cortex neoplasms; ferrets; luteinizing hormone receptors; Mustelidae; ovariectomy; orchiectomy; steroidogenesis.

The adrenal cortex is a major source of steroid hormones, which are synthesized from cholesterol through the sequential activities of a series of enzymes (Fig. 1). A dual-function enzyme, cytochrome P450 17-hydroxylase/C17-C20 lyase (P450c17), catalyzes both the 17-hydroxylation reaction required for the production of cortisol and the 17,20-lyase reaction required for the synthesis of the adrenal androgens dehydroepiandrosterone (DHEA) and androstenedione, the precursors of sex steroids.² The 17-hydroxylase and 17,20-lyase activities of P450c17 are differentially regulated; consequently, cortisol production dissociates from adrenal androgen synthesis in certain physiologic and pathophysiologic states.^1,2,8,9

View larger version (17K): [in this window] [in a new window]   Fig. 1. Steroid hormone biosynthesis in normal (solid lines) versus neoplastic (dashed lines) adrenocortical cells. All steroidogenic cells share the capacity to mobilize and cleave cholesterol. The repertoire of enzymes distal to P450scc determines the steroidogenic capacity of a given cell. Note that P450c17 has both 17-hydroxylase and 17,20-lyase activities. Cyt b5 selectively enhances the 17,20-lyase activity of P450c17 through allosteric effects. 3β-HSD = 3β-hydroxysteroid dehydrogenase, 17β-HSD = 17β-hydroxysteroid dehydrogenase, cyt b5 = cytochrome b5, DOC = deoxycorticosterone, DHEA = dehydroepiandrosterone, P450aldo = aldosterone synthase, P450c11 = cytochrome P450 11β-hydroxylase, P450c17 = cytochrome P450 17-hydroxylase/17,20-lyase, P450c21 = cytochrome P450 21-hydroxylase.

The 17,20-lyase activity of P450c17 must increase to permit biosynthesis of androgen precursors and sex steroids in gonadectomy-induced adrenocortical neoplasms of ferrets (Fig. 1). We hypothesized that increased intracellular expression of cytochrome b₅ (cyt b₅), an allosteric regulator that selectively enhances the 17,20-lyase activity of P450c17,¹ could account for the preferential production of androgenic steroids by the tumor tissue. To test this hypothesis, we screened archival specimens of adrenocortical tumors from ferrets for expression of cyt b₅.

Surgical biopsy and necropsy specimens of ferret adrenocortical neoplasms were obtained from archives of the Michigan State University Animal Health Diagnostic Laboratory (12 cases) and The Ohio State University Department of Veterinary Biosciences (16 cases). Our analysis included cases of anaplastic adrenocortical carcinoma (12), well-differentiated adrenocortical carcinoma (6), adenoma (7), and nodular hyperplasia (3). Criteria for classification of these tumors are listed elsewhere.⁶ All of these tumors were from gonadectomized ferrets with signs of AAE as documented by a review of pathology records. Several of these neoplasms contained residual normal cortex, and some had direct hepatic invasion or metastasis. For negative controls, we used autopsy specimens from 5 gonadectomized ferrets with no proliferative lesions in their adrenal glands; none of these ferrets had signs or symptoms of AAE.

Paraffin-embedded tissue sections were processed for immunoperoxidase staining as described previously.⁶ The following primary antibodies were used: 1) goat anti-mouse GATA4 immunoglobulin (Ig) G (sc-1237, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1 : 200 dilution; 2) mouse anti-human LHR hybridoma conditioned media (CRL-2685, ATCC, Manassas, VA), 1 : 100 dilution; 3) rabbit anti-human cyt b₅ (sc-33174, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1 : 200 dilution; 4) mouse anti-human inhibin- (MCA77G, Serotec, Inc., Raleigh, NC), 1 : 200 dilution; 5) goat anti-human aromatase (sc-14245, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:200 dilution; and 6) goat anti-mouse P450c17 (sc-46081, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:200 dilution. Secondary antibodies used for immunoperoxidase staining were: 1) donkey anti-goat biotinylated IgG (Jackson Immunoresearch, West Grove, PA) 1 : 200 dilution; 2) donkey anti-mouse biotinylated IgG (Jackson Immunoresearch), 1 : 200 dilution; and 3) goat anti-rabbit biotinylated IgG (NEF-813, NEN Life Science, Boston MA), 1 : 200 dilution. The avidin-biotin immunoperoxidase system (Vectastain Elite ABC Kit, Vector Laboratories, Inc., Burlingame, CA) and diaminobenzidine (Sigma-Aldrich Corp., St. Louis, MO) were used to visualize the bound antibody; slides were then counterstained with 100% hematoxylin.

There was little or no cyt b₅ immunoreactivity in normal adrenocortical tissue adjacent to neoplastic lesions (Fig. 2a, b). Nor did we observe cyt b₅ expression in adrenal tissue from gonadectomized ferrets without proliferative lesions (data not shown). This lack of cyt b₅ may account for the minimal androgen production in the adrenal glands of healthy ferrets. In contrast, cyt b₅ immunoreactivity was observed in 96% of ferret adrenocortical neoplasms, including 17 of 18 carcinomas (Figs. 2b and 3a), 7 of 7 adenomas, and 3 of 3 cases of nodular hyperplasia. The cells expressing cyt b₅ were large, lipid laden, and scattered throughout the neoplasms (Figs. 2b and 3a). Cyt b₅ staining intensity did not differ between malignant and benign adrenocortical lesions. In addition to neoplastic epithelial cells, adrenocortical tumors in ferrets often contain a spindle-cell component of uncertain clinical significance.³ Negligible cyt b₅ staining was seen in the spindle-cell component of the tumors (data not shown). All of the neoplasms that exhibited cyt b₅ immunoreactivity were from ferrets with clinical signs of AAE. Thus, cyt b₅ is a useful marker of androgen synthetic potential in these neoplasms.

View larger version (59K): [in this window] [in a new window]   Fig. 2. Adrenal gland, adrenocortical carcinoma; ferret. The junction between the zona reticularis (zr) and medulla (m) is shown. Note that normal cells in the zr lack expression of cyt b5 and GATA4, whereas, adrenocortical carcinoma (acc) cells express both of these markers of gonadal cell differentiation. Fig. 2a. HE. Fig. 2b. Cyt b5, avidin-biotin-peroxidase complex method. Fig. 2c. GATA4, avidin-biotin-peroxidase complex method. Bar = 100 µm.

View larger version (168K): [in this window] [in a new window]   Fig. 3. Adrenal gland, adrenocortical carcinoma; ferret. Note that cyt b5, LHR, aromatase, and P450c17 are coexpressed in the tumor cells. Fig. 3a. Cyt b5, avidin-biotin-peroxidase complex method. Fig. 3b. LHR, avidin-biotin-peroxidase complex method. Fig. 3c. Aromatase, avidin-biotin-peroxidase complex method. Fig. 3d. P450c17, avidin-biotin-peroxidase complex method. Bar = 50 µm.

In primates, as in ferrets, the expression of cyt b₅ in normal and neoplastic adrenocortical tissue correlates the capacity to produce androgens. The onset of cyt b₅ expression in the human adrenal cortex coincides with increases in circulating levels of adrenal androgens during adrenarche.² Similarly, induction of cyt b₅ in the adrenal cortex of female marmosets correlates with the capacity to secrete DHEA.⁵ Elevated levels of cyt b₅ were reported in adrenocortical adenomas from 2 patients with high circulating androgen levels.⁹ Conversely, functional human adrenocortical tumors that produce cortisol rather than androgens were shown to express low levels of cyt b₅.⁹

In theory, other mechanisms besides increased expression of cyt b₅ could contribute to the preferential production of sex steroids by neoplastic adrenocortical cells in neutered ferrets. Elevated LH levels stimulate cAMP production in steroidogenic cells, and cAMP-induced phosphorylation of human P450c17 stimulates its 17,20-lyase activity, whereas dephosphorylation abrogates this activity.¹⁰ In some humans with adrenocortical neoplasms, high circulating levels of DHEA or DHEA-S were attributed to reduced 3β-hydroxysteroid dehydrogenase activity in the tumors (Fig. 1).⁸ Whether these alternative mechanisms contribute to androgen production in ferret adrenocortical neoplasms awaits further study.

We concluded that cyt b₅ is upregulated during gonadectomy-induced adrenocortical neoplasia in ferrets and is a marker of androgen synthetic potential in these tumors. We propose that increased cyt b₅ expression accounts in part for the preferential production of adrenal androgens and estrogen by these neoplasms.

Acknowledgements

This work was supported by the NIH (DK075618 and DK52574), the Sigrid Juselius Foundation, and the Academy of Finland.

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