Sprague-Dawley Rat Plasma Research Articles

Biotransformation of Bisphenol AF to Its Major Glucuronide Metabolite Reduces Estrogenic Activity

Bisphenol AF (BPAF), an endocrine disrupting chemical, can induce estrogenic activity through binding to estrogen receptor (ER). However, the metabolism of BPAF in vivo and the estrogenic activity of its metabolites remain unknown. In the present study, we identified four metabolites including BPAF diglucuronide, BPAF glucuronide (BPAF-G), BPAF glucuronide dehydrated and BPAF sulfate in the urine of Sprague-Dawley (SD) rats. BPAF-G was further characterized by nuclear magnetic resonance (NMR). After treatment with a single dose of BPAF, BPAF was metabolized rapidly to BPAF-G, as detected in the plasma of SD rats. Biotransformation of BPAF to BPAF-G was confirmed with human liver microsomes (HLM), and Vmax of glucuronidation for HLM was 11.6 nmol/min/mg. We also found that BPAF glucuronidation could be mediated through several human recombinant UDP-glucuronosyltransferases (UGTs) including UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, among which UGT2B7 showed the highest efficiency of glucuronidation. To explain the biological function of BPAF biotransformation, the estrogenic activities of BPAF and BPAF-G were evaluated in ER-positive breast cancer T47D and MCF7 cells. BPAF significantly stimulates ER-regulated gene expression and cell proliferation at the dose of 100 nM and 1 μM in breast cancer cells. However, BPAF-G did not show any induction of estrogenic activity at the same dosages, implying that formation of BPAF-G is a potential host defense mechanism against BPAF. Based on our study, biotransformation of BPAF to BPAF-G can eliminate BPAF-induced estrogenic activity, which is therefore considered as reducing the potential threat to human beings.

Preparation of a Monoclonal Antibody against a Kallikrein-Like Enzyme from Agkistrodon halys pallas Venom and Its Application in a Pharmacokinetic Study

Snake venom contains bioactive materials for drug development, diagnosis, and treatment. After separating and purifying the kallikrein-like enzyme (AHP-Ka) from Agkistrodon halys pallas venom for the first time, a monoclonal antibody against AHP-Ka was prepared and characterized. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibody was developed and validated for the pharmacokinetic analysis of AHP-Ka in rat plasma. The method was calibrated using rat plasma and 1:100 dilution of plasma was selected to prepare a calibration curve to validate the precision, accuracy, and stability of the ELISA method. A good linear relationship was obtained in a working range from 3.9 ng/mL to 62.5 ng/mL with a limit of detection of 2.94 ng/mL. Intra- and inter-batch precision were less than 10%. The average recovery ranged from 94.6% to 104.4% in rat plasma at the concentrations of 5 ng/mL, 15 ng/mL, and 45 ng/mL, respectively. The ELISA method was successfully used for the pharmacokinetic study of AHP-Ka in Sprague-Dawley rat plasma after intravenous administration. The work is expected to contribute to future preclinical development of AHP-Ka.

Time-Course Effects of Intravenously Administrated Silica Nanoparticles on Blood Coagulation and Endothelial Function in Rats

Among the most used inorganic nanomaterials, silica nanoparticles (NPs) have been considered as either drug carriers or contrast agents. Though the distribution of silica NPs via the circulation appears highly probable, to date, there are few studies investigating the vascular effects of silica NPs in vivo. This study was designed specifically to investigate whether silica NPs with intravenous injection could lead to blood coagulation disorder and endothelium dysfunction in vivo. The time-course effect of silica NPs on blood coagulation, oxidative stress and the expression of soluble E-selectin (sE-selectin) and tissue factor (TF) in the plasma of Sprague Dawley (SD) rats were presented. Our data showed that a shortened prothrombin time (PT) was observed on 1 day after exposure to silica NPs, while activated partial thromboplastin time (APTT) was not affected at any time-points. After the post-injection respectively, the levels of fibrinogen (Fbg) were increased by silica NPs from 1 day to 3 days, and returned to normal value on the 7th day. Meanwhile, a sustained increase in the levels of TF and sE-selectin was elicited by silica NPs during 7 days after the injection. In addition, after 7 days of intravenously injection of silica NPs, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) in the plasma of SD rats were decreased significantly, whereas the level of malondialdehyde (MDA) was not changed obviously. In conclusion, intravenously administration of silica NPs could shorten PT but not APTT increase TF and sE-selectin release and reduce GSH-px and SOD activity in the plasma of SD rats, indicating exposure to silica NPs could early activate coagulation cascade via the extrinsic pathway, and may be dependent on endothelium dysfunction and oxidative stress.

A sensitive LC–MS/MS method for the determination of free maytansinoid DM4 concentrations—Method development, validation, and application to the nonclinical studies of antitumor agent DM4 conjugated hu-anti-Cripto MAb B3F6 (B3F6-DM4) in rats and monkeys

A sensitive, specific, and high throughput method was developed and validated for the quantitation of free maytansinoid DM4 in cynomolgus monkey and Sprague-Dawley rat plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The Sciex API 5000 mass spectrometer was operated in multiple-reaction monitoring (MRM) and electrospray positive ionization mode. An MS/MS transition of 780→216 was used to monitor the analyte DM4 and 635→547 for the internal standard (IS) ansamitocin P-3. A liquid–liquid extraction was utilized for sample pre-treatment with a volume of 100μL plasma. A Thermo Hypersil Gold PFP column was used for chromatographic separation with a 2.0min HPLC gradient. The quantitation range of the method was 0.500–100ng/mL with a lower limit of quantitation of 0.500ng/mL. The method was validated in monkey and rat plasma according to FDA guidance on Bioanalytical Method Validation (2001). The intra- and inter-day precision and accuracy were found to be within the FDA recommended acceptance criteria. The validated method was employed to monitor the free DM4 levels in plasma in the IND-enabling toxicology studies of antitumor agent DM4 conjugated hu-anti-Cripto MAb B3F6 (B3F6-DM4).

The determination of RWJ-38705 (tramadol N-oxide) and its metabolites in preclinical pharmacokinetic studies using LC–MS/MS

A rapid and reliable analytical method is described for the simultaneous determination of RWJ-38705 (tramadol N-oxide) and several of its major metabolites in the plasma of Sprague-Dawley rats and Beagle dogs. Sample preparation using solid phase extraction was followed by reversed phase liquid chromatography (LC) coupled with tandem mass spectrometric (MS/MS) detection in the positive ionization mode. The assay was linear for all analytes over concentrations ranging from approximately 6 to 2000 ng/ml. The inter-assay reproducibility was generally less than 15% while accuracy values were within 13% of theoretical. The overall recovery of the analytes ranged from approximately 40 to 64% in rat plasma and 53–75% in dog plasma. This assay has proven to be sensitive, specific and reproducible, and it has been readily implemented in preclinical PK studies. Representative plasma concentration versus time profiles resulting from administration of TNO to rats and dogs are presented in this communication.

High-performance liquid chromatographic determination of new 2,3-benzodiazepines

A simple high-performance liquid chromatographic assay with ultraviolet detection at 254 nm for simultaneous determination of 2,3-benzodiazepine derivatives (2,3-BZ2 and 2,3-BZ2Me) and their metabolites in rat plasma is described. The procedure involves a fast extraction of the drugs from the buffered sample using methanol. The extract is evaporated to dryness at 45°C and the residue is redissolved in methanol (twice). A 20-μl aliquot is injected into the liquid chromatograph and eluted with methanol–water (65:35, v/v) on a C 18 reversed-phase column. At a flow-rate of 1.5 ml/min the detection time was 3.1 min for 2,3-BZ2, 5.06 min for 2,3-BZ2Me and 10.9 min for prazepam, used as internal standard for the quantification of the studied compounds. The method has been used to investigate the steady-state concentrations of two 2,3-benzodiazepine derivatives in Sprague–Dawley rat plasma.

Agonist-Induced Release of Neuropeptide Y by Platelets

Neuropeptide Y (NPY) is a multi-functional peptide localized in a number of tissues besides the brain, and its expression is high in strains of mice with autoimmune disease. Functionally, along with its neurotransmitter role, NPY has multiple effects on both vascular tissues and tissues involved in immune responses. Previous studies demonstrated that immunoreactive NPY (i-NPY) exists in large quantities in platelets and bone marrow of some species, and suggested its release during platelet aggregation by collagen. Because NPY has been hypothesized to have a role in a number of pathophysiological states in which platelets are involved, including immune responses, hypertension and vascular smooth muscle proliferation, we sought to further characterize platelet i-NPY content and release by a diverse group of physiological platelet agonists. Platelet rich plasma and gel filtered platelets were prepared from citrated whole blood of male Sprague Dawley rats. Platelet content and concentration of i-NPY were quantified by RIA. Platelet aggregation responses (turbidometric method) and release of i-NPY were measured in rat platelet preparations stimulated by a thromboxane agonist (U44069), collagen, thrombin and ADP. All agents induced dose-dependent aggregation, although ADP and U44069 were weak agonists in citrated platelet rich plasma, ADP only inducing primary aggregation. Total i-NPY content in platelet rich plasma of Sprague-Dawley rats was 32.1±3.8 pmol/ml; more than 20 pmol/ml was released into supernatant of aggregating platelets stimulated with high dose thrombin or collagen during secondary, irreversible aggregation. Although U44069 and ADP both stimulated i-NPY release in a dose-dependent manner, release was substantially lower than with the other agonists. We conclude that i-NPY release from rat platelets is associated mainly with secondary, irreversible aggregation, and can be produced by a variety of platelet stimulating agents as part of the platelet release reaction. The present study and other recent studies demonstrate wide variability in platelet i-NPY content between and within species. Investigation of the genetic regulation of i-NPY content in platelets could offer new insights into the relationship between NPY and pathophysiology of the immune and cardiovascular systems.

Hypertension in the transgenic rat TGR(mRen-2)27 may be due to enhanced kinetics of the reaction between mouse renin and rat angiotensinogen.

The transgenic rat TGR(mRen-2)27, in which the Ren-2 mouse renin gene is transfected into the genome of the rat, develops severe hypertension with high adrenal renin and low kidney renin. These animals express both mouse and rat renin. To investigate the cause of hypertension in the TGR rat, we compared the kinetics of mouse renin acting on mouse and rat angiotensinogens. The optimum pH of the renin reaction in the Sprague-Dawley rat was 6.5, whereas the optimum pH of the reaction in the TGR rat was approximately 8.5. The optimum pH of the renin reaction in the DBA mouse was 6.0. Purified mouse Ren-2 renin acting on rat angiotensinogen showed a pH profile similar to that for the renin reaction in the TGR rat. The angiotensinogen concentration in pooled plasma from eight DBA mice was 104.5 ng angiotensin I/mL and was clearly lower than that in Sprague-Dawley rats (772.4 +/- 37.3 ng angiotensin I/mL, n = 4). The reaction of purified mouse Ren-2 renin with rat angiotensinogen was 10 times faster than with mouse angiotensinogen. Plasma renin activity in DBA mice increased dramatically on addition of rat angiotensinogen (from 253.4 +/- 66.7 to 225,000 +/- 48,000 ng angiotensin I/mL per hour). Intravenous injection of 2 or 10 microL of DBA mouse plasma into the nephrectomized Sprague-Dawley rat increased the mean arterial pressure of the rat by 27.7 +/- 4.7 and 61.8 +/- 2.7 mmHg, respectively, whereas injection of 200 microL of Sprague-Dawley rat plasma did not change the mean arterial pressure of the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic aspects of thrombus formation in mutant analbuminemic rats

To elucidate the mechanism for thrombus formation in hypoalbuminemic and hyperlipidemic subjects, the experimental thrombus formation was analyzed in Nagase analbuminemic rats (NAR). The mutant rat reveals similar biochemical findings to those of NS, such as hyperlipidemia. When thrombus was induced in a small branch of the mesenteric artery by inserting a glass micropipette, thrombus formation was observed within 4 min in NAR and Sprague-Dawley rat (SDR). In SDR, the thrombus gradually grew up in size and detached from the inserted glass micropipette within 4 min after its formation. In contrast, the thrombus formed in NAR did not dissociate from the micropipette until 10–13 min after its formation. The maximum size of the thrombus formed in NAR was about 4 times larger than that in SDR. In the presence of fibrin, the plasma samples from NAR inhibited the activity of tissue-plasminogen activator (t-PA) 1.7 times more potently than did SDR plasma. Plasma α2-plasmin inhibitor activity was significantly higher than that in SDR. Albumin significantly enhanced the t-PA-catalyzed activation of plasminogen, suggesting that the serum albumin might contribute, at least in part, to the plasma fibrinolytic activity. Thus, the higher thrombogenic potential in NAR than in SDR might be due to the reduced thrombolytic activity in NAR.

Purification and characterization of two kinds of low molecular weight kininogens from rat (non-inflamed) plasma. One resistant and the second sensitive to rat glandular kallikreins.

Two kinds of low molecular weight kininogens (identified as A and B) were isolated from pooled plasma of Sprague-Dawley rats. They show a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol, and the molecular weights are 68,000 for low Mr kininogen A and 73,000 for low Mr kininogen B. Although the molecular weights and amino acid compositions of the low Mr kininogens are similar, rat submaxillary and urinary kallikreins released bradykinin from low Mr kininogen B, whereas low Mr kininogen A was resistant to these enzymes. The COOH-terminal portion of low Mr kininogen A was isolated after cyanogen bromide treatment, and the amino acid sequence of the COOH-terminal 55 residues including the T-kinin (Ile-Ser-bradykinin) was determined. The COOH-terminal portion consists of two sequences with substitution of 4 residues. One peptide corresponds to alpha 1-major acute phase protein (Cole, T., Inglis, A. S., Roxburgh, C. M., Howlett, G. J., and Schreiber, G. (1985) FEBS Lett. 182, 57-61) and the other to the TI-kininogen predicted from a cDNA study (Furuto-Kato, S., Matsumoto, A., Kitamura, N., and Nakanishi, S. (1985) J. Biol. Chem. 260, 12054-12059). The results demonstrate that there exist at least two kinds of low Mr kininogens with clearly different function in rat plasma: one of them, low Mr kininogen A, is a precursor of T-kinin and is resistant to kallikreins, and the second, low Mr kininogen B, is sensitive to tissue kallikreins and shares properties with bovine and human low Mr kininogens. The results also demonstrate that T-kininogen is a mixture of two isoproteins which correspond to alpha 1-major acute phase protein or TI-kininogen, respectively. We could not detect the low Mr kininogen corresponding to the TII-kininogen predicted from the cDNA study of Furuto-Kato et al.

Production and release of plasminogen by isolated perfused rat liver.

In view of conflicting evidence for a major hepatic role in the synthesis of circulating plasminogen, the precursor of the fibrinolytic enzyme plasmin, we carried out the present study with a sensitive assay in order to measure the accumulation of small quantities of plasminogen in a recycling rat liver perfusion system. We have purified plasminogen from Sprague-Dawley rat plasma and have raised a monospecific antiserum against it in rabbits. Isolated rat liver perfusions were performed with an oxygenated recycling perfusate consisting of a perfluorotributylamine/Pluronic F-68 emulsion (Fluosol 43) free of plasma proteins and blood cells. The system was shown to be capable of synthesizing albumin and transferrin. The cumulative appearance of plasminogen in the perfusate was measured by a sensitive, specific radioimmunoassay. Plasminogen concentration increased progressively during the first 2 hr of perfusion; the observed average net synthesis in five separate experiments was approximately 35 microgram/hr per 100 g of body weight. Exposure of the perfused liver to 18 microM cycloheximide inhibited additional increase in the titer of plasminogen. Evidence for de novo synthesis was provided by the incorporation of 14C-labeled leucine into specific immunoprecipitates of plasminogen and the inhibition of this incorporation by cycloheximide. Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a single peak of radioactivity corresponding to Mr of 82,000. These data indicate that the liver is a major site of plasminogen production.

Aflatoxicol: major aflatoxin B1 metabolite in rat plasma.

Aflatoxicol, a carcinogenic metabolite of the foodborne carcinogen aflatoxin B1 previously known only as a bioreduction product in vitro, was identified as the major aflatoxin metabolite in the plasma of Sprague-Dawley rats, a susceptible species, that had been doses orally or intravenously with aflatoxin B1 labeled with carbon-14. Alfatoxicol, however, was not detected in the plasma of similarly dosed mice and monkeys, which are both resistant to aflatoxin B1-induced cardinogenesis. The formation of aflatoxicol both in vitro and in vivo may be an indicatory of species sensitivity to aflatoxin-induced carcinogenesis and may be useful in the prediction of human susceptibility.