Effect of zinc on androgen metabolism in the human hyperplastic prostate.

17B-Hydroxy-5a-androstan-3-one (dihydrotestosterone) may be the active androgen required for maintenance of many androgen-dependent tissues (King & Mainwaring, 1974). In the human prostate gland, testosterone is converted into dihydrotestosterone by aNADPH-dependent A4-3-oxosteroid 5a-oxidoreductase (5a-reductase) (Ofner et al., 1970). This enzyme has been located in both nuclear and microsomal fractions from human prostatic tissue, and found to be sensitive to thiol-group-blocking reagents and low concentrations of bivalent cations, including zinc (Wallace & Grant, 1975). In the human male, zinc may play a general role in sexual development (Sandstead et al., 1967; Caggiano et al., 1969). The human prostate contains a relatively high concentration of zinc (Schrodt et al., 1964), probably held in storage as a component of prostatic secretion (Mackenzie et al., 1962). A zinc-binding protein, isolated from human prostatic tissue by Reed & Stitch (1973), may be involved in this process. In the rat, a connection exists between prostatic zinc and androgen status (Millar et al., 1957). A study was therefore made of the relationship between androgen metabolism and zinc in the human prostate. Surgically removed hyperplastic prostatic tissue was used at once or after storage at -70°C. Tissue (log) was minced and washed with 0.15M-NaCI. The NaCl wash was termed the extracellular fraction, but also contained cell debris and blood. The mince was homogenized by using an Ultra-Turrax and a Potter-type homogenizer, and cellular componentswereseparated by a method similar to that of Kowarski et al. (1969). The nuclear fraction was further freed from cytoplasmic material by centrifugation through 2.0~-sucrose at 96OOOgfor 2h. For 5a-reductase measurement, 2ng of [1,2-jH]testosterone (specific radioactivity 14Ci/mmoI), O . ~ ~ M N A D P H and 40m-nicotinamide were incubated in 1 ml of Tris buffer (lOmM, pH7.0) containing 5m~-MgCl, and 50m~-NaCI. [3H]Testosterone, [3H]dihydrotestosterone and, in some cases, [3H]5aandrostane-3aJ 7jI-dio1, were determined after ensuring radiochemical purity by derivative formation followed by chromatography on thin layers of alumina in cyclohexaneethyl acetate (4: 1, v/v). Inhibition of 5a-reductaseactivity by theaddition ofO.1 mM-ZnC1, to bothnuclear and microsomal fractions could bereversed by 1 mwdithiothreitol, EDTA, o-phenanthroline and, to some extent, by high concentrations of citrate. Lineweaver-Burk analysis of the kinetics of zinc inhibition indicates that, in both nuclear and microsomal fractions, zinc inhibits in a competitive-like manner with respect to cofactor, but in a noncompetitive-like manner with respect to substrate. This suggests that this cation may bond at or near the NADPH-binding site. Fig. 1 shows a double-reciprocal plot for the inhibition observed with 0.1 mM-ZnC1, on the oxidation of NADPH by the microsomal 5 a-reductase. Zinc was measured by atomic-absorption spectrophotometry after dry ashing of prostatic tissue preparations. It appeared to be concentrated in the extracellular, nuclear and cytosol fractions. Whole-tissue zinc was also measured in 13 hyperplastic prostates at two sites within each gland. There were wide variations in concentrations within any one gland, and the mean of all measurements was 1210+901 (s.D.),ugof zinc/gdry wt.,