Mouse Urine Samples Research Articles

264. Non-Invasive Monitoring of the Response of Orthotopic Bladder Tumors to GM-CSF Gene Therapy Using the Prostate Specific Antigen Gene as a Reporter

Our purpose was to determine the feasibility and accuracy of modifying the murine bladder cancer cell line MB49 to secrete the prostate specific antigen (PSA) as a biomarker for monitoring orthotopic bladder tumor growth and to further evaluate the effectiveness of GM-CSF gene therapy in this model. The modified MB49-PSA cells behaved similarly to the parental cell line and produced high levels of PSA both in vitro and in vivo. In a subcutaneous model the level of PSA produced correlated with tumor volume (r=0.96). To test the model, MB49-PSA cells were implanted orthotopically into C57BL/6 mice. On the fourth day after implantation, small tumors measuring as small as 0.2–0.3mm in diameter were identified and these were sufficient to produce measurable PSA in both serum and urine. Mice with confirmed tumors were given liposome-mediated GM-CSF gene therapy on the fifth day after implantation. Therapy was administrated twice a week for 3 weeks intravesically. Tumor growth and response to treatment was monitored at 2–3 day intervals by measuring PSA level in mouse serum and urine samples by ELISA. Measurement of serum PSA level over the treatment period showed that tumor growth was inhibited by GM-CSF gene therapy. Up to 50% of the treated mice were cured. Cytokine array analysis revealed that GM-CSF gene therapy appeared to act by inducing the production of other cytokines and chemokines. Our results demonstrate that the PSA marker protein is constitutively expressed by MB49-PSA cells implanted in mice and changes in its serum levels reflect the tumor burden in vivo. Our results also indicated that liposome-mediated GM-CSF gene therapy is effective in treating murine bladder cancer. The study provides a simple, reliable and non-invasive method to evaluate therapeutic modalities such as gene therapy for bladder cancer Figure 1.

Analysis of urinary metabolites of tea catechins by liquid chromatography/electrospray ionization mass spectrometry.

Tea has been proposed to have beneficial health effects which have been attributed to the polyphenolic compounds known as catechins. The bioavailability and biotransformation of these compounds, however, are not clearly understood. In this study, we used liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) to determine urinary glucuronidated and sulfated tea catechins and their metabolites (including methylated and ring-fission metabolites) based on the detection of deprotonated molecular ions and aglycone fragment ions. The compound resolution was achieved both chromatographically and mass spectroscopically. After green tea administration, the major conjugates appeared in human, mouse, and rat urine samples were identified as monoglucuronides and monosulfates of (-)-epigallocatechin (EGC) and (-)-epicatechin. We also found O-methyl-EGC-O-glucuronides and -O-sulfates and O-methyl-epicatechin-O-sulfates in human urine. (-)-5-(3',4',5'-Trihydroxyphenyl)-gamma-valerolactone (M4) and (-)-5-(3',4'-dihydroxyphenyl)-gamma-valerolactone (M6), the ring-fission metabolites of EGC and (-)-epicatechin, respectively, were also predominantly in monoglucuronide and monosulfate forms in the urine. In comparison to rats, the urinary metabolite profiles of tea catechins in mice resemble more closely to those in humans. This is the first report describing direct simultaneous analysis of multiple tea catechin conjugates in urine samples. This method will allow more thorough investigations of the biotransformation of tea polyphenols.

Pharmacokinetics of ethylene glycol. II. Tissue distribution, dose-dependent elimination, and identification of urinary metabolites following single intravenous, peroral or percutaneous doses in female Sprague-Dawley rats and CD-1 mice.

1. [1,2]-14C-Ethylene glycol (EG) was given to female CD (Sprague-Dawley) rats and CD-1 mice in order to determine tissue distribution and metabolic fate after intravenous (iv), peroral (po), and percutaneous (pc) doses. Rats were given doses of 10 or 1000 mg/kg by each route, and additional pc doses of 400, 600 or 800 mg/kg. Mice were also given iv and po doses of 10 or 1000 mg/kg, and intermediate po doses of 100, 200 or 400 mg/kg. Mice were given po doses of 100 or 1000 mg/kg, and both species were given a 50% (w/w) aqueous po dose to simulate antifreeze exposure. 2. For both species, EG is very rapidly and almost completely adsorbed after po doses. Perorally administered EG doses produced similar dose-dependent relationships described in prior studies for the disposition and excretion of iv doses. 3. The tissue distribution of EG following either iv or po routes was essentially the same, with similar percentages recovered for each dose by both routes and for either species. 4. Cutaneously-applied EG was slowly and rather poorly adsorbed in both species, in comparison with po-dose administration, and urinalysis after undiluted po doses indicated that EG probably penetrates rat skin in the parent form. There was an absence in both species of dose-dependent changes in disposition and elimination following the pc application of EG. 5. 14C-labelled EG, glycolic acid and/or oxalic acid accounted for the majority of the detectable radioactivity in the urine samples from all dose routes in the rat, while glycoaldehyde and glyoxylic acid were not detected in any of the urine fractions evaluated. Similar increases in glycolate production with increasing dose were also observed in mouse urine samples from iv and po dosing. Also, glyoxylate and oxalate were absent from mouse urine. 6. Oxidative metabolic pathways appeared to be saturated at high po doses in both species, resulting in a shift from principally 14CO2 exhalation to urinary 14C excretion, while the onset of capacity-limited metabolic changes appears to occur at lower doses for mice than for rats. 7. In summary, rats and mice displayed several similarities in the manner in which low doses of EG by several routes are distributed, metabolized, and excreted, but the onset of capacity-limited changes in metabolism occurs at lower doses for mice than for rats. Such differences in the disposition of EG may provide important interpretive information to help explain differences observed in developmental toxicity and nephrotoxic responses between these two rodent species.

MHC-Associated and MHC-lndependent Urinary Chemosignals in Mice Are Expressed via the Hematopoietic System

The change in genetically determined urine odors which appears after experimental bone marrow transplantations in mice was examined in order to test whether the HMC is the only group of genes that influences the chemosensory identity via the hematopoietic system. 5 female rats were trained in a computer-controlled olfactometer to discriminate urine odors of two MHC congenic or background congenic inbred strains of mice. Transfer-of-training tests with urine samples of irradiated mice which were restored with bone marrow either from an MHC congenic or a background congenic inbred strain reveal a change of urinary chemosignals after both types of experimental bone marrow transplantation. Thus, both MHC-associated as well as MHC-independent urinary chemosignals are expressed via the hematopoietic system.

Variations in mouse (Mus musculus) urinary volatiles during different periods of pregnancy and lactation

Mouse urine samples from different pregnancy and lactation periods were examined by capillary gas chromatography to assess variations in the volatile signals that may affect the endocrine function of other females. Statistically significant changes in the excretion of certain urinary volatiles were observed; from 26 readily quantifiable constituents, 14 appear to be under the endocrine control. These selected components, positively identified through mass spectrometry and retention data, and the synthetic standards are ketones, unsaturated alcohols, esters, and cyclic vinyl ethers.

Atmospheric pressure ionization mass spectrometry: the routine determination of 2,4,5-trichlorophenoxyacetic acid and its metabolites.

This study demonstrated the utility of negative ion atmospheric pressure ionization mass spectrometry for the routine quantitation of 2,4,5-trichlorophenoxyacetic acid and its glycine and taurine amide metabolites in mouse blood, urine and feces samples. The quantitation of 2,4,5-trichlorophenoxyacetic acid in blood follows a short cleanup procedure and used 2,4,5-trichlorophenoxy-13C6-acetic acid as the stable label isotope diluent. A more extensive cleanup procedure utilizing high-pressure liquid chromatography was required for the determination of 2,4,5-trichlorophenoxyacetic acid and its two metabolites in urine and feces. The glycine amide metabolite was quantitated by the 13C stable isotope diluent method. The taurine amide relied on an initial separation step followed by using a second 2,4,5-trichlorophenoxy-13C6-acetic acid spike fot its isotope diluent. Alkaline hydrolysis of the metabolites, followed by methylation, allowed the methyl ester of 2,4,5-trichlorophenoxyacetic acid to be solely used as the analyte in the negative ion atmospheric pressure ionization mass spectrometry quantitation step.

Evaluation of adrenal function in mice by measurement of urinary excretion of free corticoids

Methods for the determination of urinary total free corticoids and urinary free corticosterone in small laboratory rodents are described. Total free eorticoids and free corticosterone were determined in samples of 50–200 μl urine by a competitive protein binding method using human plasma transcortin as the source of binding protein. For both methods employed the reliability criteria were satisfactory. The validity of both methods was tested by measuring both total free corticoids and free corticosterone in urine samples of mice with the obese-hyperglycemic syndrome (gene symbol C57BL/6J-ob/ob) and their lean controls (gene symbol C57B1/6J). Measurements were taken from urine samples under basal conditions and after adrenal stimulation or suppression. Urinary corticoid levels were significantly different between the two mouse lines, 15 ng/day for the lean and 75 ng/day for the obese mice, reflecting plasma corticosterone levels. After stimulation by ACTH urinary corticoids rose 3–5-fold whereas suppression by dexamethasone was followed by a decrease to very low levels. The contribution of corticosterone to total corticoids averaged 50% in lean and 30% in obese mice. It is concluded that urinary total excretion of free corticoids as well as urinary excretion of free corticosterone offer a valuable parameter of adrenal function in mice. Furthermore, when small laboratory rodents are used, under many circumstances measurements in urine are more advantageous than measurements in plasma.