CHEMICALS AND DRUGS
Carrageenan and 17β-hydroxyesradiol were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), Oestrone, 4-hydroxyestradiol, 4-hydroxyestrone, 16α-hydroxyestradiol, 16α-hydroxyestrone, 2-methoxyestradiol, 2-methodxyestradiol, 4-methoxyestrone, and 4-methoxyestradiol were purchased from Steroids Inc. (Newport, RI, USA). Urethane was purchased from Biobasic Inc. (Toronto, Canada). B-Glucuronidase/Arylsulphatase (Helix pomatia, Type HP-2, ≥500 Sigma units β-glucuronidase and ≤37.5 units sulfatase activity) and dansyl chloride (Dns-Cl) 98% HPLC grade were purchased from Sigma-Aldrich Chemical Co. All other chemicals were of the highest available analytical grade.
ANIMALS AND TREATMENT
Male Sprague–Dawley rats (300 g weight) were acclimatized in the animal house facility of King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia, for at least 1 week before experimentation. Animals were kept at 20 ± 2°C and 65 ± 10% relative humidity for the duration of the experiment. Standard food pellets and water were supplied ad libitum. All experiments were performed between 8:00 and 10:00 a.m. Animal handling and treatment was approved by the bioethical and research committee of King Abdulaziz University.
Experimental prostatitis was induced in rats as previously described with minor modifications . Briefly, animals were anaesthetized by i.p. injection of urethane (1 g/kg). A surgical V-shape incision was made in the supra pubic region in the rats, exposing the prostate gland. A volume of 50 µL of carrageenan solution (2%) was slowly injected into the ventral prostate and the wound was then surgically sutured and dressed with sterile povidone iodine solution. Sham operations were performed in animals of control groups without carrageenan injection. Animals were divided into four groups (n= 6). The control group was subjected to sham operation; the oestrogen-alone group was subjected to sham operation and received 17β-oestradiol in DMSO (5 mg/kg) via i.p. injection 3 h before being killed; the prostatitis group was subjected to intraprostatic carrageenan injection; and the oestrogen and prostatitis group was subjected to intraprostatic carrageenan injection and received 17β-oestradiol (5 mg/kg) via i.p. injection 3 h before being killed. Oestradiol treatment dose and duration were based on pilot experiments as well as the work of Cavalieri et al. . All animals were allowed free access to food and water for 48 h after surgery then were killed by cervical dislocation. Prostatic tissues were collected post mortem and aliquots were distributed and snap frozen at −80°C until further analysis; portions of the prostatic tissues were fixed in buffered formalin solution (4%) for histological assessment.
To assess the inflammatory status of the ventral prostate lobe, PGE2 and TNF-α within ventral prostatic tissue were quantified. Accurately weighted prostatic tissues were ice crushed in liquid nitrogen and then homogenized for 15 min in PBS (pH 7.4). The amounts of PGE2 and TNF-α were determined using Quantikine® rat immunoassay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Concentrations of PGE2 and TNF-α were normalized based on protein concentration.
To assess the expression of oestrogen-metabolizing enzymes, total RNA isolation from ventral prostatic tissues was performed using RNeasy Mini Kit® (Qiagen Inc. Valencia, CA, USA). Reverse transcription was undertaken to construct a cDNA library from different treatments using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). PCR reactions were then performed using a Taq PCR Master Mix Kit (Qiagen Inc.) and amplification products were assayed using QIAxcel System® capillary electrophoresis (Applied Biosystems). Primer sequences were as follows: aromatase forward primer GCT-TCT-CAT-CGC-AGA-GTA-TCC-GG and reverse primer CAA-GGG-TAA-ATT-CAT-TGG-GCT-TGG; catechol-O-methyltransferase (COMT) forward primer ATC-TTC-ACG-GGG-TTT-CAG-TG and reverse primer GAG-CTG-CTG-GGG-ACA-GTA-AG; nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1 (NQO-1) forward primer CAG-GGT-CCT-TTC-CAG-AAT-AAG and reverse primer CTG-GTT-GTC-GGC-TGG-AAT-GGA-C; rat steroid sulphatase forward primer TAA-CCC-AGG-GAC-AAC-CTC-TG and reverse primer GGT-GTA-GCC-TTG-ACC-CTT-GA; 17 β-hydroxysteroid dehydrogenase type-I forward primer AGT-GCT-CAT-TAC-CGG-TTG-CT and reverse primer CTT-GCT-CAT-AAC-CAC-GCT-GA; 17 β-hydroxysteroid dehydrogenase type-II forward primer TTC-TCT-GCA-AAG-CCT-GGA-GT and reverse primer GGC-TCC-GAA-GAA-GTT-CAC-TG. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference housekeeping gene with forward primer AAA-CCC-ATC-ACC-ATC-TTC-CA and reverse primer GTG-GTT-CAC-ACC-CAT-CAC-AA.
Oestrogen metabolites were assessed in ventral prostatic tissues using the liquid chromatography mass spectrometry (LC-MS) method as described previously, with minor modifications . On the day of analysis, samples were ice crushed in liquid nitrogen, homogenized and enzymatically hydrolysed using β-glucuronidase/arylsulphatase in 1 mL of 0.1 m acetate buffer (pH 5.5) for 24 h. Steroid content was extracted using 5 mL dichloromethane (three times). The combined organic layer was subjected to evaporation under a weak stream of nitrogen gas and the dried residue was completely dissolved in 100 µL Dns-Cl (1 mg/mL in acetone) and 100 µL 0.1 m sodium bicarbonate (pH 9.3). After shaking for 2 s, samples were heated at 60 °C for 5 min, cooled, and a volume of 20 uL was injected for LC-MS analysis using mixture reaction monitoring (MRM) mode. An Agilent 1200 series HPLC with Ion Trap 6320 MS/MS detector was used (Agilent Technology, Santa Clara, CA, USA). The analysis was performed on Agilent Zorbax Extend C18 column (150 mm × 46 mm, 5 microns) with a guard column Extend C18 (2.2 × 2.2 mm) at 35°C. The HPLC system was programmed to clean column with 100% acetonitrile for 10 min, followed by 100% water for 10 min, and allowed to equilibrate for 10 min with the mobile system. The ion-Trap detection was set as follows: MRM mode was selected and the M + H+ for Dns-Oestrogen were selected, range 450–850 m/z, target mass 500 m/z, max accumulation time 200 ms, ramp range 4500–1500 v. Peaks were verified by m/z and retention time.
Reference standard calibration curves (n= 3) of each oestrogen metabolite in similar biomatrix were conducted and validated for accuracy and precision over a concentration range of 0.1–90 ng/mL.
Histological and immunohistochemical analysis for ventral prostatic tissues were performed according the laboratory routine protocol. Briefly, paraformaldhyde fixed tissues were embedded in paraffin wax. Cross-vertical sections (5 µm) were obtained and, after dewaxing and rehydration, sections were stained with haematoxylin and eosin (H&E).
Paraffin-embedded ventral prostate tissues were further used for immunohistochemical staining of cyclin-D1, ER-α and ER-β. Briefly, cross-vertical sections (5 µm) were obtained and, after dewaxing and rehydration, sections were incubated with 3% H2O2 for 30 min to eliminate the endogenous peroxidase activity. Non-specific binding sites were blocked with normal donkey serum for 30 min and then incubated overnight at 4°C in mouse antiserum (Abcam Inc., Cambridge, UK) against cyclin-D1 (dilution 1:100), ER-α (dilution 1:25) or ER-β (dilution 1:100). After rinsing in PBS, sections were incubated in peroxidase-conjugated donkey anti-mouse IgG (dilution 1:200; Jackson ImmunoResearch Lab, Inc. West Grove, PA, USA) for 1 h. For colouration, PBS-washed sections were incubated with a mixture of 0.05% 3,3’-diaminobenzidine containing 0.01% H2O2 at room temperature until a brown colour was visible, and then washed with PBS, counterstained with H&E, and mounted. Percent DAB-positive cells per high power field were counted and used for quantitative comparison . Rat uterus samples were used as quality controls for ER immunostaining.
Data are presented as mean (sem). anova with a least significant difference post-hoc test was used to determine significance using SPSS® for Windows, version 17.0.0. A P value of <0.05 was considered to indicate statistical significance.