Spectrophotometric Method for the Assay of Steroid 5α-Reductase Activity of Rat Liver and Prostate Microsomes

Transcription

1 ANALYTICAL SCIENCES APRIL 2013, VOL The Japan Society for Analytical Chemistry Spectrophotometric Method for the Assay of Steroid 5α-Reductase Activity of Rat Liver and Prostate Microsomes Atsushi IWAI,* 1 Teruki YOSHIMURA,* 2 Keiji WADA,* 2 Satoshi WATABE,* 3 Yuki SAKAMOTO,* 4 Etsuro ITO,* 4 and Toshiaki MIURA* 1 *1 Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita, Sapporo , Japan *2 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido , Japan *3 BL Co., Ltd., Numazu, Shizuoka , Japan *4 Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Sanuki , Japan A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17β-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-nad) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-nadh having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR. (Received January 15, 2013; Accepted February 26, 2013; Published April 10, 2013) Introduction The steroid 5α-reductase (5α-SR, EC ) metabolizes testosterone to 5α-dihydrotestosterone (5α-DHT), which is a more potent androgen than testosterone and exerts its function in androgen-responsive tissues. 1 The metabolite 5α-DHT is responsible for benign prostate hyperplasia (BPH) and prostate cancer (PCa), major neoplastic diseases in older men. 2,3 There are three isoforms of 5α-SR, the isozyme type 1 (5α-SR1), type 2 (5α-SR2) and type 3 (5α-SR3). Isozymes 5α-SR1 and 5α-SR2 have been well characterized. 1,4 Type 5α-SR1, the major isozyme in skin and liver, has a broad basic ph optimum and low affinity for testosterone (K m = 1 5 μm) while 5α-SR2, the major isozyme in prostate, has an acidic ph optimum and high affinity for testosterone (K m = 4 50 nm). Both the isozymes are overexpressed in BPH and PCa. 5 7 Recently, 5α-SR3 has been identified, which is overexpressed in the tissues of hormone-refractory prostate cancers. 8 Since accumulated evidence suggests that 5α-SR inhibitors such as finasteride are useful for the treatment of BPH and PCa, a simple assay method for 5α-SR is desirable for screening 5α-SR inhibitor. 3,9 12 The methods available for the assay of 5α-SR in biological samples is limited to radioisotopic methods, namely involving (1) incubation of radiolabeled testosterone with To whom correspondence should be addressed. miura@pharm.hokudai.ac.jp NADPH and enzyme sources of 5α-SR, (2) extraction of steroids with organic solvent, and (3) separation of the metabolites from testosterone by TLC 4 or HPLC 5 followed by measurement of their radioactivities. These methods are sensitive enough to assay 5α-SR in biological samples, but require special facilities and equipment to handle radioactive compounds and entail tedious procedures. Recently, non-radioisotopic assay methods for 5α-SR using LC-MS have been developed, 13,14 but they are time-consuming because the solid-phase extraction procedure 13 or extraction with organic solvent 14 are needed prior to LC-MS analysis. We describe here a simple spectrophotometric method for the assay of 5α-SR in microsomes of rat liver and prostate, which is based on the sensitive determination of 5α-reduced metabolites, 5α-DHT and 5α-androstane-3α,17β-diol (5α-diol), by enzymatic cycling using 3α-hydroxysteroid dehydrogenase (3α-HSD, EC ) in the presence of excess NADH and thionicotinamide-adenine dinucleotide (thio-nad). Experimental Materials and equipment Recombinant 3α-HSD from Comamonas testosteroni expressed in Escherichia coli (3α-HSD-E, 80 U mg 1 ) was obtained from Kikkoman Corp. (Japan). 5α-DHT and 5α-diol were purchased from Sigma and Steraloids (Newport, RI), respectively. Thio-NAD, NADH and NADPH were obtained

2 456 ANALYTICAL SCIENCES APRIL 2013, VOL. 29 from Oriental Yeast (Japan). Finasteride was donated by Asuka Pharmaceutical Co. (Japan). All other chemicals were of analytical grade. Solutions of testosterone, 5α-DHT, 5α-diol and finasteride were prepared by dissolving in ethanol followed by dilution with appropriate buffer. The 3α-HSD was dissolved in a 1:1 mixture of 0.1 M Tris HCl buffer (ph 7.5) and glycerol and stored at 20 C before use. NADH, thio-nad and NADPH solutions were prepared by dissolving in 0.1 M phosphate buffer (ph 8.0), 10 mm glycine buffer (ph 4.0) and 50 mm Tris HCl buffer (ph 9.0), respectively. Absorption measurements were made with a Shimadzu UV-1600 spectrophotometer using a 2.5-mm diameter cuvette thermostated at 37 C. Preparation of rat liver and prostate microsomes All animal experiments were performed according to the protocols approved by the Animal Care and Use Committee of Tokushima Bunri University. Wistar strain male rats (10 weeks old) were decapitated and whole livers and prostate were immediately removed. The livers were minced and homogenized in three volumes of ice-cold 3 mm Tris buffer (ph 7.4) containing 1.15% KCl and 0.1 mm EDTA. The homogenate was sequentially centrifuged at 9000g for 20 min and g for 60 min at 4 C. The resulting pellets were resuspended in 10 mm phosphate buffer (ph 7.4) and used as liver microsomes. The prostate microsomes were prepared according to the method of Mitamura et al. 13 Briefly, minced prostate was homogenized in three volumes of ice-cold 40 mm phosphate buffer (ph 6.5) containing 0.3 M sucrose and 1 mm dithiothreitol (DTT) and centrifuged at 1500g for 20 min. The supernatant was centrifuged at g for 60 min at 4 C. The collecting pellets were resuspended in 40 mm phosphate buffer (ph 7.5) containing 0.3 M sucrose and 1 mm DTT and used as prostate microsomes. The microsomes were stored at 80 C before use. The protein content of the microsomes was measured by the method of Lowry et al. 15 The liver and prostate microsomes were used after dilution with 40 mm phosphate buffer of ph 7.0 or ph 5.5 containing 0.3 M sucrose and 1 mm DTT for the assay of liver 5α-SR1 and prostate 5α-SR2, respectively. Enzymatic cycling of 5α-DHT or 5α-diol Enzymatic cycling was carried out at 37 C in the incubation mixture (1.0 ml) of system A or B. System A contained 1.5 mm thio-nad, 0.5 mm NADH, 20 U ml 1 3α-HSD and 5α-DHT or 5α-diol in 0.1 M potassium phosphate buffer (ph 8.0). System B contained 1.5 mm thio-nad, 0.3 mm NADH, 20 U ml 1 3α-HSD and 5α-DHT or 5α-diol in 0.1 M Tri HCl buffer (ph 8.5). The reaction was initiated by adding 3α-HSD (50 μl) and the increase in absorbance at 400 nm was measured from 1 to 4 min after initiating the reaction (ΔA 1 4 min). The number of cycles per min (cycling rate) occurring between 5α-DHT and 5α-diol was estimated from ΔA 1 4 min and the molar absorptivity of thio-nadh (11900 mol 1 l cm 1 ). 16 Assays of 5α-SR activity of rat liver and prostate microsomes and inhibitory activity of finasteride Rat liver microsomal 5α-SR1-catalyzed reduction of testosterone was carried out at 37 C for 30 min in 200 μl of 40 mm phosphate buffer (ph 7.0) containing 60 μm testosterone, 800 μm NADPH and ng ml 1 mirosomal protein. The reaction was started by adding the microsomes (20 μl) and stopped by heating at 80 C for 5 min. After cooling to room temperature, a portion (100 μl) of the reaction mixture was transferred to a cuvette, 850 μl of the cycling reagent (0.1 M potassium phosphate buffer, ph 8.0, containing 1.76 mm thio-nad and 0.6 mm NADH) was added and the mixture was preheated at 37 C for 3 min. Enzymatic cycling was initiated by adding 50 μl of 400 U ml 1 3α-HSD and ΔA 1 4 min at 400 nm was measured. As a control, the liver microsomes were used after heating at 80 C for 5 min. Calculations of 5α-SR1 activity were determined from the ΔA 1 4 min and calibration curve (Fig. 2). One unit (U) of the activity was expressed as the sum (μmol) of 5α-DHT and 5α-diol formed from testosterone per minute at 37 C. Rat prostate 5α-SR2 activity was assayed by incubating 300 μl of 40 mm phosphate buffer (ph 5.5) containing 250 nm testosterone, 800 μm NADPH and μg ml 1 prostate microsomal protein at 37 C for 30 min. The reaction was started by adding the microsomes (24 μl) and stopped by heating the reaction mixture at 80 C for 5 min. After cooling to room temperature, the mixture was centrifuged at 5000g for 2 min to remove precipitates. A portion (250 μl) of the supernatant was transferred to a cuvette, supplemented with 700 μl of the cycling reagent (0.1 M Tris HCl buffer, ph 8.5, containing 2.0 mm thio-nad and 0.4 mm NADH) and preheated at 37 C for 3 min. Enzymatic cycling was initiated by adding 50 μl of 400 U ml 1 3α-HSD and 5α-SR2 activity was calculated as described above. In inhibitory experiments, the liver 5α-SR1 and prostate 5α-SR2 activities in the presence of finasteride (final concentration: nm) were measured in the same manner described above at microsomal protein concentrations of 1.12 and 620 μg ml 1, respectively. Results and Discussion Determination of the sum of 5α-DHT and 5α-diol by enzymatic cycling For the assay of 5α-SR in microsomes, both 5α-DHT and 5α-diol have to be determined as 5α-reduced metabolites of testosterone because 5α-DHT is further metabolized to 5α-diol by mammalian liver microsomal 3α-HSD in the presence of NADPH. 17 In order to develop a simple assay method for the sum of 5α-DHT and 5α-diol at the picomole level, we employed enzymatic cycling using 3α-HSD, which catalyzes the oxidoreduction of 3α-hydroxysteroids. The assay principle is depicted in Fig. 1. Because 3α-HSD utilizes both NAD(H) and thio-nad(h) as cofactors, 16 it catalyzes the substrate cycling between 5α-DHT and 5α-diol in the presence of excess thio-nad and NADH. In each turn of the cycle, one molecule of thio-nad is reduced to thio-nadh which can be measured from its absorbance at 400 nm with no interference from excess NADH having an absorption maximum at 340 nm. The accumulated thio-nadh is proportional to the sum of 5α-DHT and 5α-diol. Similar substrate cycling using 3α-HSD has been successfully applied to the assay of serum bile acids. 18,19 Therefore, we performed a series of experiments to optimize the cycling conditions with 10 nm 5α-DHT at 37 C. From the results of these experiments, we established two incubation systems using 0.1 M phosphate buffer of ph 8.0 (system A) and 0.1 M Tris HCl buffer of ph 8.5 (system B). System A, which contained 0.5 mm NADH, 1.5 mm thio-nad and 20 U ml 1 3α-HSD, gave a cycling rate of 600 cycle per min and showed a linear relationship (R 2 = 0.999) between increased absorbance at 400 nm (ΔA 1 4 min) and concentration of 5α-DHT in the range of 1 30 nm. The same results were obtained for 5α-diol. On the other hand, system B contained 0.3 mm NADH, 1.5 mm thio-nad and 20 U ml 1 3α-HSD. Compared with system A,

3 ANALYTICAL SCIENCES APRIL 2013, VOL Fig. 1 Assay principle for 5α-SR. Fig. 2 Calibration curve of system A for 5α-DHT in the presence of excess testosterone (600 nm) and NADPH (80 μm). Values are expressed as the mean ± SD for six determinations. Assay conditions are described in the Experimental section. a higher cycling rate of 800 cycle/min was obtained with system B though the linear response range was limited to nm of 5α-DHT (R 2 = 0.998). Figure 2 shows the calibration curve of system A for 5α-DHT in the presence of excess testosterone and NADPH, which are used in the assay of 5α-SR. The relative standard deviation at 5 nm was 2.6% (n = 6). Because Comamonas testosteroni 3α-HSD used in this study does not utilize NADP(H) as a cofactor and testosterone as a substrate, 20 neither NADPH nor testosterone interfered with the measurement of 5α-DHT and 5α-diol. Assays of 5α-SR activity of rat liver and prostate microsomes Since the present enzymatic cycling was shown to be applicable to the direct assay of the sum of 5α-DHT and 5α-diol, we employed this enzymatic cycling to the assay of rat liver and prostate 5α-SR. The liver 5α-SR1 catalyzed reduction of testosterone was carried out at its optimal ph of 7.0 1,4 using 40 mm phosphate buffer (ph 7.0) containing 0.3 M sucrose and 1 mm DTT. After incubation of testosterone (6 μm) with NADPH (800 μm) and liver microsomes at 37 C for 30 min, the reaction was stopped by heating at 80 C for 5 min. The sum of 5α-DHT and 5α-diol formed was then measured by diluting the reaction mixture with the cycling reagents to give the enzymatic cycling conditions of system A. Because of the high efficiency of the enzymatic cycling and high activity of 5α-SR1 in liver microsomes, the formation of 5α-reduced metabolites was detected with a small amount of the microsomal protein. The formation of 5α-reduced metabolites with 672 ng ml 1 of microsomal protein was linear with respect to an incubation Fig. 3 Linearity between the rate of metabolite (5α-DHT + 5α-diol) formation and concentration of microsomal protein. Values are expressed as the mean ± SD for three determinations. (A) Liver microsomes, (B) prostate microsomes. Assay conditions are described in the Experimental section. time up to 60 min. A linear calibration curve (R 2 = 0.993) was obtained for the protein concentration of ng ml 1 (Fig. 3A). The 5α-SR1 activity of rat liver microsomes used was calculated to be 4.3 nmol min 1 per mg of protein. On the other hand, more than 50 μg ml 1 of microsomal protein was used for the assay of prostate microsomal 5α-SR2 because of its low activity. The prostate 5α-SR2 was assayed by incubating with testosterone (250 nm) and NADPH (800 μm) at 37 C for 30 min in 40 mm phosphate buffer of ph 5.5 optimal for 5α-SR2. 1,4 After the reaction was stopped by heating at 80 C for 5 min, the reaction mixture was centrifuged at 10000g for 2 min to remove precipitates. A portion of supernatant was then diluted with the cycling reagents to give the enzymatic cycling conditions of system B. The formation of 5α-reduced metabolites with 496 μg ml 1 of

4 458 ANALYTICAL SCIENCES APRIL 2013, VOL. 29 separation procedures. The method was based on 3α-HSD-catalyzed enzymatic cycling between 5α-DHT and 5α-diol, the metabolites formed in the NADPH-dependent reduction of testosterone with the enzyme sources of 5α-SR, in the presence of excess thio-nad and NADH. Because of high sensitivity and no interference from testosterone, the method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride. The present method can be effectively applied on a routine basis to assay a large number of samples for the screening of 5α-SR inhibitors. Acknowledgements Fig. 4 Inhibition of 5α-SR isozymes by finasteride. Liver 5α-SR1 activity was assayed with 6 μm testosterone and liver microsomal protein (1.12 μg ml 1 ) at ph 7.0. Prostate 5α-SR2 activity was assayed with 250 nm testosterone and prostate microsomal protein (620 μg ml 1 ) at ph 5.5. Assay conditions are described in the Experimental section. The study was supported by the Knowledge Cluster Initiative from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by SENTAN, Japan Science and Technology Agency. We also express our thanks to Asuka Pharmaceutical Co. for supplying the finasteride. microsomal protein was linear with respect to incubation time up to 40 min. Figure 3B shows the calibration curve (R 2 = 0.999) for the microsomal protein. The 5α-SR2 activity of the prostate microsomes used was estimated to be 1.6 pmol min 1 per mg of protein. Assays of inhibitory activity of finasteride In order to develop the treatment for 5α-DHT associated diseases such as BPH and PCa, a simple test system has been required to screen for 5α-SR inhibitors Because of its simplicity and high sensitivity, the present method is also applicable to the simple test system for screening 5α-SR inhibitors. After confirming that finasteride showed no effect on the enzyme cycling of 5α-DHT, we performed the inhibition experiments by finasteride of rat liver and prostate microsomal 5α-SR (Fig. 4). From the results, the concentrations of finasteride required to inhibit 5α-SR activity by 50% (IC 50) were estimated to be 21 nm for liver 5α-SR and 20 nm for prostate 5α-SR, respectively. The inhibitions of rat 5α-SR1 and 5α-SR2 by fenasteride have been investigated by using COS cells transiently expressing 5α-SR1 and 5α-SR2. 21 The IC 50 values of finasteride to 5α-SR1 and 5α-SR2 were evaluated to be 23 and 5.2 nm respectively in whole cell assay, whereas those were 13 and 1.0 nm respectively in the assay with crude enzyme preparations. 21 The IC 50 value of finasteride to rat 5α-SR in prostate microsomes was also evaluated to be 11 nm by Häusler et al., nm by Igarashi et al. 23 and 237 nm by Mitamura et al. 13 The reported IC 50 values of fenasteride to rat 5α-SR in prostate homogenate were in the range from 6.8 to 147 nm The reason for such a difference may be related to differences in experimental conditions of enzyme activity evaluation such as ph, testosterone concentration and enzyme preparation. 24 Conclusions The methods available for the assay of 5α-SR in biological samples are limited to the radioisotopic method and LC-MS analysis. 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