Study On Genistein's Pharmacokinetics And Its Mechanism Of Transport And Metabolism

AIM: (1) To study the different pharmacokinetics of genistein in female and male rats. (2) To investigate the mechanism of the genistein in the rat liver microsomes. (3) To investigate accumalation and transport of the genistein in the Caco-2 cells. (4) To provid index in the research of pharmacology and toxicology of phytoestrogens.METHODS: (1) Genistein(9mg-kg"1) was orally administered to rats. The blood samples were collected at 0.08, 0.17, 0.33, 0.67, 1, 1.33,2, 4, 8,12, 24h. At the same time, organs of female and male rats were collected at 0.6667, 1.3333, 8h after dosing. The genistein in plasma and organs were extracted twice by vortexing with 2.0mL mixture of methyl tert tubtyl ether and pentane (v/v=8:2). The organic phase was removed into the tubes and then evaporated in ventilation cabinet. The genistein and glucuronidated genistein in plasma and in different organs were detected by RP-HPLC. (2) SD rat liver microsomes were prepared by using ultracentrifuge method. The incubation mixture consisted of 0.15mol ·L~-1 KCl-0.1mol ·L~-1 PBS(pH7.4), NADP (0.5mmol ·L~-1 ) generating system, the genistein (100μmol ·L~-1 ) and microsomal proten(1mg·mL~-1 ) in a final volume of lmL. After being incubated for different time(5~60min) at 37℃, the reaction was terminated by -30 ℃ refrigerator. The RP-HPLC was used to determine the concentration of genistein in the incubation mixture; The genistein(100μmol ·L~-1 ) wereincubated in different amount of microsomal protein(0.5~21mgmL~-1) at 37℃ for 30min, the concentration of the genistein in the incubation mixture was determined by the RP-HPLC; The different concentration of genistein (1×10~-7-1.6×10~-3mol·L~-1) were incubated with 1mg·mL~-1 microsomal protein at 37℃ for 30min, and the RP-HPLC was used to determine the concentration of the genistein in the incubation mixture; The different concentration of daidzein(1 ×10~-7~1.6×10~-3 mol·L~-1) were incubated with 1mg·mL~-1 microsomal protein at 37℃ for 30min, and the RP-HPLC was used to determine the concentration of the daidzein in the incubation mixture. The Michaelis-Menten parameters (Michaelis constant [Km] and maximal velocity [Vmax]) in SD rat liver microsomes were determined. The values of Km and Vmax were initially estimated by analyzing Eadie-Hofstee plot. (3) The genistein(100μmol·L~-1) were incubated with microsomal protein (lmg-mL" ) and different kind of selective CYP inhibitor at 37℃ for 30min in order to investigate its effect on the metabolism of the drug. After incubation, the RP-HPLC was used to assay the concentration of the genistein in the incubation mixture. (4) SD rat liver microsomes were prepared from male or female rats. The optimized system of enzyme catalytic reaction of genistein in rat liver microsomes was set up. The reaction velocity of genistein in female and male rats liver microsomal protein (lmg-mL"1) were assessed by incubating genistein(100μmol·L~-1) with CYP1A2 antibody or selective CYP1A2 inhibitor furafylline, and the percentage of the relative activity of CYP1A2 were derivated by using the date of the reaction velocity. (5) Caco-2 cells were grown in 24 well culture plates using growth medium at an initial seeding density of 5000/cm2 . The cells were used 21~25 days after seeding. The genistein (50μmol·L~-1) was added in HBSS for various time up to 120 min at 37℃ and 4℃. Cells were washd 3×in ice cold PBS before storing at -80℃ freeze thawing three times, splitting cells. The RP-HPLC was used todetermine the concentration of the genistein in cell lysate. Protein concentrations for all aspects of this study determined using a Bradfold method on DU-800 spectrophotometer; The different concentration of genistein(10, 50, 100, 200, 400umol-L"1) in HBSS were incubated with Caco-2 cells at 37°C and 4°C for 20min, splitting cells. The RP-HPLC was used to determine the concentration of the genistein in the cell lysate. (6) Cell grown on the base of 24 well plates after an overnight incubation with various P-glycoprotein or MRP inhibitors followed by genistein (SOumol-L"1) at 37°C for 20min. The RP-HPLC was used to determine the concentration of the genistein in the cell lysate. (7) The genistein(50j^mol-L'1) was added in HBSS for various time up to 120 min at 37°C and 4°C, splitting cells. The cell lysate(0.4mL) and 0.2mL /3-glucuronidase solution(800U-mL"1 in 0.17mol-L"' ammonium acetate, pH4.6) were added into the tube, and they were incubated in 37 °C water bath for 24h. The RP-HPLC was used to determine the concentration of the genistein in cell lysate. (8) The daidzein^OOumol-L"1 , 250umol-L"1) in HBSS was incubation with Caco-2 cells for 30min and followed by genistenXSOumol-L"1) at 37°C for 20min. The RP-HPLC was used to determine the concentration of the genistein in the cell lysate. (9) The sodium azide(10 mmol-L"1, 50 mmol-L"1) and DNP (O.Smmol-L"1 , lmmol-L"1) in HBSS was incubation with Caco-2 cells for 30min and followed by genistein^Oumol-L"1) at 37 °C for 20min. The RP-HPLC was used to determine the concentration of the genistein in the cell lysate. (10)The omeprazole (lOumol-L"1, 25umol-L"1 and 50umol-L"') in HBSS was incubation with Caco-2 cells for 30min and followed by genistehXSOumol-L"1) at 37 °C for 20min. The RP-HPLC was used to determine the concentration of the genistein in the cell lysate. (11) Caco-2 cells were grown on Millcell using growth medium at an initial seeding density of 5000/cm2. The cell were used 21-25 days after seeding. Transportin expressed as ng/cm2. The genistein(lOjiM) was added to Ap chamber . Sample was removed from the Ap and Bsa chamber at 0.67, 1.5, 3.5, 5.5, 7.5, 9.5h. The RP-HPLC was used to determine the concentration of the genistein and glucuronidated genistein in the HBSS; The genistein(lOuM) was added to Ap and Bsa chamber respectively. Sample was removed from the Ap and Bsa chamber at 5, 10, 20, 40, 90min. The RP-HPLC was used to determine the concentration of the genistein in the HBSS; Caco-2 cell grown on the Millcell after an overnight incubation with verapami^lOOumol-L"1). Then the genistein(lOuM) was added to Ap chamber. Sample was removed from the Bsa chamber at 0.0833, 0.1667, 0.3333, 0.6667, 1.5h. The RP-HPLC was used to determine the concentration of the genistein in the HBSS. RESULTS : (1) The concentration of genistein in ovaries or uterus was significantly higher than that in testis, and there were no remarkable differences of genistein in other organs between female and male rats. The AUC were 1.12 (ug-h-mL"1) in female and 0.59 (ug-h-mL"1) in male, and Cmax were 0.17 (ug-mL"1) in female and 0.11 (ug-mL"1) in male. The T1/2 were 5.77h in females and 4.49h in males. The concentration of glucuronidated genistein in female rats was much higher than that in male rats. (2) The elimmation of genistein in microsomal protein was increased linearly with the increase of incubation time (0~10min). The reaction velocity of genistein got to steady state at 30min. With the increase of microsomal protein(0.5~2mg-mL"1) at 37°C for 30min, the reaction velocity of genistein was increased linearly. The reaction velocity of genistein and daidzein were increased with the substrate concentration(lxlO"7~l><10"4mol-L*1) at 37°C for 30min. The Km and Vmax of genistein were 54.33 + 4.94 and 1.84+0.12. The Km and Vmax of daidzein were 100.74 + 7.79 and 3.66 + 0.22. The Km and Vmax of daidzein were significant larger comparing with*genistein(P<0.05). (3) The result showed that the metabolism of genistein wassignificantly inhibited by furafylline in SD rat liver microsomes. While quinidine, sulfaphenazole, tranylcypromine diethyldithiocarbamate and ketoconazole showed little inhibitory effect on the metabolism of genistein. (4) The metabolism of genistein in male and female microsomes was inhibited by CYP1A2 antibody (1:400) after incubation for 30min. The percentage of the control activity of microsomes in male and female rat were (20.95+2.13%) and (13.73 + 1.26%) respectively( PO.01). The metabolism of genistein in male and female microsomes was inhibited by selective CYP1A2 inhibitor furafylline p.^SumoM/1). The percentage of the control activity of microsomes in male and female rat were (58.02 + 3.35%) and (43.82+2.65%) respectively( PO.01). (5) The uptake of Coca-2 cells on genistein was taken on increase at 37°C and 4°C with the increaseing of incubation concentration 10, 50, 100, 200, 400umol-L"1) of genistein. The amount of genistein was accumulated by Coca-2 cells at 37°C was significantly larger than at 4°C(P<0.01). The uptake of the genistein was taken on linear increase at 37 °C with the increasing of incubation time (0~10min). The time of maximal accumulation was lOmin and the concentration of maximal accumulation was 2.61+0.07 nmol-mg-I/'prot"1. The uptake of the genistein was taken on linear increase at 4°C. The time of maximal accumulation was 40min and the concentration of maximal accumulation wasl.26 + 0.09 nmol-mg-L~1prot~1. (6) After a 24h incubation with Verapamil^Oumol-L"1, lOOumol-L"1 )and cyclosporin A (50umol-L"', lOOumol-L"1) followed by genistein(50umol-L"1), the result showed that the amount of genistein in Caco-2 with Verapamil and cyclosporin A was significantly larger than that of control(P<0.05). But after incubation with erythromycin(50umol-L"1, lOOumol-L'1) , the genistein in Caco-2 was no difference with control. (7) After lOmin incubation with genistein at 37°Cthere were glucuronidatedgenistein in Caco-2 cells. But there are no glucuronidated genistein in Caco-2 at 4 °C . (8) After an 24h incubation with daidzein(100umol-L"1, 250umol-L"1), the accumulation of genistein by Caco-2 was striking higher than that of control at 37°C for 20min(P<0.01). (9) After a 30min incubation with sodium azide(10 mmol-L"1, 50 mmol-L"1) and DNP (O.Smmol-L"1 , lmmol-L"1), the accumulation of genistein in Caco-2 was significantly lower than that of control at 37°C for 20min(P<0.05). (10) The result showed that the accumulation of genistein in Caco-2 cells was not significantly effected by omeprazole (lOumol-L"1 -. 25umol-L~1and SOumol-L"1) at 37°C for 20min. (11) Transport of genistein through Caco-2 monolayers occurred in both apical to basolateral and basolateral to apical directions. However, basolateral to apical transport was of a greater rate than transport in the other direction(P<0.01); the results showed the rate of transport of genistein(10umol-L"') from the apical to basolateral with verapamil (lOOumol-L"1) was higher than that of control (PO.01) ;When transport of genistein^Oumol-L"1) in the apical to basolateral , we can determin the glucuronidated genistein in apica chamber only.CONCLUSION: (1) The distribution of genistein in sexual organ of female and male rats was strikingly different. Compared with male rats, the absorption of genistein and glucuronidation of genistein were much more in female rats, and this resulted in the prolongation of the half-life of genistein. (2) It was shown that daidzein was the most suitable substrate of rat liver microsomes. (3) CYP1A2 was involved in metabolism of genistein. In terms of metabolism, CYP1A2 inhibitors may interact with genistein, this could reduce the rate of genistein metabolism. (4) The results indicated that the activity of male rats liver CYP1A2 was higher than that of the female rat. (5) There are active transport and passive diffusion in accumulation of genistein in Caco-2 cells. (6) The genistein efflux were inhibited by verapamil and...