Tail Vein Of Rats Research Articles

Ultrasound-mediated microbubble destruction enhances VEGF gene delivery to the infarcted myocardium in rats

Objective To investigate the possibility of improving the delivery of vascular endothelial growth factor (VEGF) gene to the myocardium in rats by using ultrasound-mediated microbubble destruction (UMMD). Methods Fifteen male Wistar rats underwent left anterior descending coronary artery ligation in this study. The rats were divided into three groups 3 days after ligation. Ultrasound microbubble vectors (UMVs) attaching to pcD2VEGF121 gene were injected into the tail vein of rats with or without simultaneous echocardiographic microbubble destruction in two groups. The third group was used as control group. VEGF protein expression and formation of new blood vessels were evaluated by immunohistochemical technique during autopsy on 15 rats at 2 weeks after gene transformation. Microvascular density (MVD) in the area with myocardial infarction was counted under a microscope. Results VEGF protein expression and MVD in the ischemic myocardium were higher in the rats receiving UMMD than in the group that did not receive UMMD. Conclusion UMMD is a noninvasive method to effectively improve the delivery of targeted genes to the heart.

Intercellular adhension molecule-1 in the pathogenesis of heroin-induced acute lung injury in rats

The expression of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of heroin-induced acute lung injury (ALI) in rats was investigated. The model of ALI was established by intravenous injection of heroin into tail vein in rats. Thirty-six rats were randomly divided into heroin-treated groups (1 h, 2 h, 4 h, 6 h and 24 h) and normal control group. Changes in histopathologic morphology and biological markers of ALI were measured. The expression of ICAM-1 in lung tissue was detected by using immunohistochemistry and RT-PCR. The results showed that the W/D ratio and protein contents in BALF of the heroin-treated groups were significantly higher than that of the, control group (P<0.01). The histopathological changes in the lung tissue were more obvious in heroin-treated groups. The ICAM-1 protein and mRNA expression in the lung tissue of heroin-treated groups were significantly increased as compared with that of the control group (P<0.01), and correlated with the ALI parameters in a time-dependent manner. Increasing of ICAM-1 expression was involved in the formation of heroin-induced lung injury. Furthermore, the level of expression was positively correlated with the severity of lung injury.

Relationship between endothelin and the pathophysiology of tissue factor-induced and lipopolysaccharide-induced disseminated intravascular coagulation in rats: a study examining the effect of an endothelin receptor antagonist.

We investigated the relationship between endothelin, a potent vasoconstrictor peptide, and the pathophysiology of disseminated intravascular coagulation (DIC), using two models of DIC. Experimental DIC was induced by sustained infusion of 50 mg/kg lipopolysaccharide (LPS), or 3.75 U/kg thromboplastin, for 4 h via the rat tail vein. The effect of administration of a non-selective endothelin receptor antagonist (TAK-044) (2, 10, or 50 mg/kg, from -0.5 to 4 h) on thromboplastin-induced DIC was not significant. However, LPS-induced elevation of alanine aminotransferase, creatinine and glomerular fibrin deposition was significantly suppressed by co-administration of TAK-044 in a dose-dependent manner, although no effect of TAK-044 was observed on the platelet count, fibrinogen concentration or the level of thrombin-antithrombin complex. Moreover, plasma levels of D-dimer, which reflect the grade of fibrinolysis of cross-linked fibrin, were significantly increased by co-administration of each dose of TAK-044 in the LPS-induced DIC model in rats. Our results suggest that vasoconstriction, as well as depressed fibrinolysis, contribute to severe organ dysfunction in LPS-induced, but not thromboplastin-induced, DIC, and that endothelin plays a role in the development of organ injury in LPS-induced DIC in rats.

In Vivo Evaluation of the Boronated Porphyrin TABP-1 in U-87 MG Intracerebral Human Glioblastoma Xenografts

Boron neutron capture therapy (BNCT) is an adjuvant therapy that has the potential to control local tumor growth. A selective delivery of sufficient amounts of boron to individual tumor cells, compared to surrounding normal tissues, is the key for successful BNCT. We have designed and synthesized a new highly water-soluble boronated porphyrin, TABP-1, as a possible BNCT agent. When we injected the maximum tolerated dose (MTD: 15 mg/kg) of TABP-1 systemically into the tail vein of athymic rats bearing intracerebral (i.c.) human glioblastoma U-87 MG xenografts, the compound accumulated preferentially in brain tumors compared to normal brain; however, the level of boron in the tumors was less than the 30 microg/g of tissue that is generally considered necessary for BNCT. We next investigated whether convection-enhanced delivery (CED) could improve the boron distribution. The compound was administered directly into i.c. tumors using an osmotic minipump attached to a brain-infusion cannula. TABP-1 doses from 0.25 to 1.0 mg infused locally over 24 h produced tumor boron concentrations greater than those obtained by systemic administration at the MTD. For example, CED administration of 0.5 mg of TABP-1 produced a tumor boron level of 65.4 microg/g of tumor, whereas the serum level was only 0.41 microg/g (tumor to serum ratio of approximately 160:1). CED also produced relatively high tumor to normal brain ratios of approximately 5:1 for ipsilateral brain and approximately 26:1 for contralateral brain tissues at the 0.5 mg dose. Thus, we may be able to achieve therapeutic BNCT efficacy with minimal systemic toxicity or radiation-induced damage to normal tissue by administering TABP-1 using CED.

PENGARUH PRAPERLAKUAN JUS PISANG AMBON ( MUSA PARADISIACA L. ) TERHADAP BIOAVAILABILITAS SULFAMEZATIN PADA TIKUS PUTIH JANTAN

The influence of ambon banana’s fruit ( Musa paradisiaca L. ) juice on the bioavailability of sulfamezathin on the rat that aim to know there are interaction result from their consumption together has been studied. The study was using experiment’s animal is white rats Wistar’s spesies that have age around 3-4 months and their weight 200-300 g. This experimental was used completely randomized design with three group’s test, each group’s test is contain seven rats. The first group is pretreatment single dose sulfamezathin (60 mg/kg ww; peroral ), second is sullfamezathin and banana ( 12,6 g/kg ww; peroral ) together and third group is sulfamezathin after one hour dose banana like second group. Respectively blood samples take up from tail vein of rat at 15, 30, 45, 60, 90, 120, 150, 180, 240, 360, 480 and 600 minutes. Consentrations of Sulfamezatin were measured use Bratton Marshall methode with spektrofotometer instrument at 545 mn wavelength. Consentrations of sulfamezathin versus times by software of Stripe used to calculate pharmacokinetics parameters in each group’s test and the value parameters of pharmacokinetics have statistic analysis by ANOVA and if there are difference’s significant ( p Bioavailability, Sulfamezathin, Ambon banana, Musa paradisiaca L.

Effect of antisense TGF-beta1 oligodeoxynucleotides in streptozotocin- induced diabetic rat kidney.

Transforming growth factor (TGF)-β1 is an important fibrogenic factor that is involved in the pathogenesis of diabetic nephropathy. We evaluated the effect of circular antisense TGF-β1 oligodeoxynucleotides (ODNs) on the TGF-β1 expression in the rat mesangial cell culture and in streptozotocin (STZ)-induced diabetic rats. Circular antisense TGF-β1 ODNs were found to be stable in rat serum, significantly decreasing TGF-β1 mRNA expression compared with linear antisense ODNs in the rat mesangial cell culture. Circular antisense TGF-β1 ODNs were introduced into the tail vein of normal rats using hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer method and were confirmed to be delivered effectively into the kidney, liver, lungs, and spleen. To inhibit the overexpression of TGF-β1 in diabetic kidneys, we introduced circular antisense TGF-β1 ODNs into the STZ-induced diabetic rats. On day 13 after circular antisense TGF-β1 ODNs injection, TGF-β1 mRNA and protein expression markedly decreased and urinary TGF-β1 excretion rate also dropped in the circular antisense TGF-β1 ODNs-treated diabetic rats. These results suggest that circular antisense TGF-β1 ODNs may be a useful tool for developing new therapeutic application for progressive diabetic nephropathy.

Three‐color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood

Experiments described herein were designed to evaluate the performance characteristics of a flow cytometry-based system that scores the incidence of peripheral blood micronucleated reticulocytes (MN-RETs). These procedures represent the continued refinement of a previously reported anti-CD71-based method (Dertinger et al. [1996]: Mutat Res 371:283-292), with the following modifications: incorporation of a third fluorescent label to exclude platelets from the MN-RET region, and use of a CD71-associated fluorescence thresholding technique to increase data acquisition rates. Mouse, rat, and human blood samples were analyzed using both the previously described two-color procedure (anti-CD71-FITC and propidium iodide) and a newly developed three-color technique (which adds an antiplatelet-PE antibody). The rodent specimens were also evaluated by standard microscopy procedures (acridine orange staining). Mouse blood was collected via heart puncture of vehicle- and 5-fluorouracil-treated CD-1 mice; blood samples from saline-treated Sprague-Dawley rats were collected from the tail vein and via heart puncture. Rodent blood samples were analyzed by both the two- and three-color methods. Human blood specimens, obtained via arm venipuncture from cancer patients undergoing radiation therapy, were analyzed for MN-RETs using the two-color method. Subsequently, blood samples from a single chemotherapy patient were analyzed by both the two- and three-color methods. Finally, the chemotherapy patient blood samples and blood samples from 15 healthy volunteers were evaluated at very high densities in conjunction with a CD71-associated fluorescence thresholding technique. Results of these investigations showed that data from mouse blood analyzed by the two- and three-color procedures correlated well with microscopy data (r values = 0.917 and 0.937 for the two- and three-color methods, respectively); all three methods confirmed the genotoxicity of 5-FU. Data from rat tail vein samples showed improved reproducibility with the three-color technique, but no significant difference between the two techniques was seen with the heart puncture specimens. Human blood analyzed according to the two-color procedure produced unreliable results, as platelets and platelet aggregates impacted the rare MN-RET scoring region. The three-color technique effectively overcame this problem and produced reproducible measurements that fell within expected ranges. For human blood analyses, the high cell density/CD71-thresholding technique provided significant improvements over the low-density technique, as it allowed data acquisition to occur approximately six times faster with no loss of sensitivity.

Biodistribution and anti-tumor efficacy of doxorubicin loaded glycol-chitosan nanoaggregates by EPR effect

An in vivo tumor targeting test of glycol-chitosan nanoaggregates was carried out with FITC-conjugated glycol-chitosan nanoaggregates (FTC-GC) and the doxorubicin conjugated glycol-chitosan (GC-DOX). To investigate its biodistribution in tumor-bearing rats, glycol-chitosan was labeled with fluorescein isothiocyanate (FITC), which formed nanoaggregates with a diameter of about 250 nm in aqueous media. GC-DOX nanoaggregates containing acid-sensitive spacers were prepared. The GC-DOX formed micelle-like nanoaggregates spontaneously in aqueous media. GC-DOX nanoaggregates had a narrow and unimodal size distribution, and its hydrodynamic diameter measured by dynamic light scattering ranged from 250 to 300 nm. A loading content of doxorubicin into GC-DOX nanoaggregates as high as 38%, with 97% loading efficiency, could be obtained using a physical entrapment method. A tumor-bearing animal model was developed by inoculating tumor cells into the back of a rat. The FTC-GC nanoaggregates were injected into the tail vein of tumor-bearing rats and their tissue distribution was examined. The FTC-GC nanoaggregates were distributed mainly in kidney, tumor and the liver and were scarcely observed in other tissues. They were maintained at a high level for 8 days and their distribution in tumor tissues increased gradually. This suggests that chitosan nanoaggregates accumulate passively in the tumor tissue due to the enhanced permeability and retention (EPR) effect. Doxorubicin loaded GC-DOX nanoaggregates (DOX/GC-DOX) were injected into the tail vein of tumor-bearing rats and their anti-tumor effect was examined. Tumor growth was suppressed over 10 days.

The effect of aerial parts of Echinacea on the circulating white cell levels and selected immune functions of the aging male Sprague–Dawley rat

Echinacea is one of the most widely used over-the-counter herbal preparations that purport to “improve immune system function”, especially when taken as a short course of therapy (6–8 weeks). Since many purchasers are older individuals, a double-blind, placebo-controlled study was performed to investigate whether Echinacea could affect total and differential white cell counts, phagocytic activity and interleukin (IL-2) levels in 12-month-old, healthy, male Sprague–Dawley rats when administered over an 8-week period. Echinacea (50 mg/kg of aerial parts) mixed with peanut butter or peanut butter alone was fed to 16 rats, which were receiving regular food and water ad libitum. Cell counts and immune functions were determined on rat tail vein blood on a weekly basis. Echinacea significantly increased circulating total white cell counts during the first 2 weeks of administration, and IL-2 levels during the final 5 weeks of the study period ( p<0.05). Differential counts were altered during the entire 8-week study, with mononuclear cells significantly increased to the detriment of granulocytes ( p<0.05). No such changes were observed in animals given peanut butter alone. No difference was observed in phagocytic function between animals given Echinacea or peanut butter alone. These studies suggest that aerial components of Echinacea affect both mononuclear cell levels and circulating IL-2 levels in older animals.

Effects of osteoporotic cytokines in ovary-intact and ovariectomised rats with induced hyperthyroidism; is skeletal responsiveness to thyroid hormone altered in estrogen deficiency?

This experimental study was designed to examine the effects of hyperthyroidism on osteoporotic cytokines such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha in the physiological concentrations and in the deficiency of estrogen. We investigated the effects of thyroid hormones on cytokines and bone metabolism in L-thyroxine induced ovary-intact and ovariectomised rats, as levels of cytokines were increased in hyperthyroidism. The rats were divided into three groups. In the first group, L-thyroxine-induced hyperthyroid rats were ovariectomised (OVX), while the OVX rats were administered L-thyroxine in the second group. The third group received sham-operation. Blood samples taken from the tail vein of rats were analyzed for plasma T3, T4, TSH and serum IL-1beta, IL-6, TNFalpha, calcium (Ca), phosphorous (P), parathyroid hormone (PTH), alkaline phosphatase (ALP), bone-specific alkaline phosphatase (b-ALP). L-thyroxine administration increased the cytokines, ALP and b-ALP and decreased PTH, while there was no change in Ca and P. However, the ovariectomy of these rats did not change the levels of cytokines, Ca, P, PTH, ALP, and b-ALP. In ovariectomised rats, the cytokines, ALP and b-ALP increased but not Ca and P conversely, PTH decreased. L-thyroxine administration to ovariectomised rats did not change the levels of cytokines, Ca, P, PTH, ALP and b-ALP. In sham-operated rats there was no change in any of the parameters compared with initial values. Thyroid hormones may not be effective on bone metabolism in estrogen deficiency.

Marked neuropeptide Y-induced contractions via NPY-Y 1 receptor and its desensitization in rat veins

The aim of this study was to investigate neuropeptide Y (NPY)-induced vasoconstrictions in rat blood vessels and which NPY receptor subtype is involved in vasoconstrictions. NPY produced marked contractions in rat common jugular, brachial, portal, femoral and tail veins, and vena cava inferior, whereas it produced little or no contractions in rat common carotid, brachial, femoral and tail arteries, and thoracic and abdominal aortae. The maximal NPY-induced contractions were larger than maximal phenylephrine (PE)-induced contractions in the veins. These NPY-induced contractions were blocked by the Y 1 antagonists, SRL-21, and BIBP3226 but not by the Y 5 antagonist, L-152804. A Y 2 agonist, NPY (13–36), did not produce contractions. RT-PCR showed that NPY-Y 1 was the only receptor subtype in the veins indicating that NPY-induced contractions are mediated through the Y 1 receptor. Pretreatment with NPY showed a rapid and long-lasting desensitization of these contractions. The marked NPY-induced contractions and its desensitization in the veins suggest the physiological relevance of NPY in the venous circulation.

Effect of Oral Adsorbent (AST-120) on Renal Function, Acquired Renal Cysts and Aortic Calcification in Rats with Adriamycin Nephropathy

Aims: The effect of oral adsorbent, AST-120, on the experimental renal disease induced by adriamycin, uninephrectomy and high protein diet proposed as a model of acquired cystic disease of the kidney was investigated. Methods: 3 mg of adriamycin was injected into the tail vein of rats and 4 weeks later right-side nephrectomy was performed, 2 weeks thereafter 26 rats with urinary protein excretion between 100 and 358 mg/day were selected from 60 rats. Two groups, 13 rats in each group, namely the AST-120-treated group and control group, both of which had equal renal damage before the administration of AST-120 or placebo. AST-120 (0.4 g/100 g BW/day) was administered for 19 weeks. Results: Serum creatinine and BUN in the AST-120-treated group were significantly lower (serum creatinine: 3.3 ± 2.1 vs. 7.1 ± 2.7 mg/dl, p < 0.003) and creatinine clearance was higher (0.62 ± 0.49 vs. 0.29 ± 0.30 ml/min, p < 0.05) at the final examination than in the control group. Survival rate which was examined using another set of 9 rats was higher in AST-120-treated rats than in AST-120-untreated rats. Serum indoxyl sulfate was significantly lower at all times after using AST-120 in the AST-120-treated group than in contrast to the control group. Histological examination revealed less severe interstitial and cystic changes in the AST-120-treated group. This suggests that AST-120 can prevent or retard the development of acquired renal cystic disease in this model. Aortic calcification tended to be less severe in the AST-120-treated group because of less serum Ca × P products. Conclusion: The AST-120-treated group significantly decreased serum creatinine and increased creatinine clearance with less severe renal cystic changes in this model during the later weeks of administration of AST-120 or at death, accompanied with the tendency of less severe aortic calcification.

Effects of sexual intercourse on testicular function

Objective: We evaluated the effects of sexual intercourse on testicular function in fertile and infertile rats. Design: Markers of Leydig and Sertoli cell secretory functions were evaluated prior to and during sexual intercourse in fertile and infertile rats. Materials/Methods: Each one of four-month-old-Wistar male rats of groups A1 (n=10; fertile rats), A2 (n=10; fertile rats), B1 (n=10; bilaterally cryptorchid infertile rats), and B2 (n=10; bilaterally cryptorchid infertile rats) was placed at the same cage with 2 female rats in oestrus. Immediately after the entrance of the male rat into the cage all the males of groups A1 and B1 were killed. Intratesticular testosterone profiles (ITPs), testicular transferrin profiles and androgen binding protein profiles (ABPPs) in testicular cytosols were evaluated. Rats of groups A2 and B2 were allowed to have a sexual intercourse. Immediately after the first ejaculation (first postejaculatory interval) rats of groups A2 and B2 were killed and ITPs, testicular transferrin levels, and ABPPs were evaluated. Prior to sacrificing each rat of groups A1, A2, B1 and B2 blood was collected from the tail vein of this male rat and the serum levels of FSH, LH, testosterone and prolactin were evaluated. Detected spermatozoa in the vaginae of the females confirmed the performance of complete intercourses. Ten and 12 additional four-month-old fertile male Wistar rats (group C and group D, respectively)were allowed to have sexual intercourse with female rats in oestus (one male was placed in the same cage with two females). However, at the time of intercourse the males of group C were disturbed by visual and audial stimuli. ITPs and ABPPs were evaluated immediately after the first ejaculation in all rats of group C. Rats of group D were killed during intromissions prior to ejaculation. ITPs and ABPPs were evaluated in each rat of group D. Results: ITPs were significantly larger (p <0.05; Wilcoxon’s test) in the group A2 (136 ± 13 ng/g testis) than in group A1 (114 ± 10 ng/g testis) and in the group B2 (91 ± 11 ng/g testis) than in group B1 (73 ± 7 ng/g testis). In contrast there were no significant differences in serum FSH, LH, testosterone and prolactin levels and in testicular ABPPs and transferrin concentrations between groups A1 and A2, and between groups B1 and B2. ITPs and testicular ABPPs were significantly smaller in groups C and D than in group A2. The latter parameters were significantly larger in groups C and D than in group A1. Conclusions: Sexual intercourse results in an increase in the ITPs in both fertile and infertile subjects. In contrast sexual intercourse does not affect Sertoli cell secretory function. This increase in ITPs appears not to be due to an enhanced pituitary gonadotropin secretion and is partially inhibited when the sexual intercourse is interrupted or the male is disturbed during sexual intercourse. Whether multiple sexual intercourses in infertile subjects increasing ITPs have a positive influence in spermatogenesis needs to be evaluated. Supported by: Research Grant from MFC Clinic, Yonago, Japan.

Preferential uptake of zinc, manganese, and rubidium in rat brain tumor

The search for radionuclides that can be an index of viability in proliferating cells is important for nuclear medicine diagnosis of brain tumors. On the basis of the finding that 65Zn is preferentially taken up in rat brain tumors, the uptake of various radionuclides was examined in rat brain tumor by using the multitracer technique. Male Fisher rats were intrahippocampally injected with C6 glioma cells. Fourteen days after implantation, the radioactive multitracer solution was injected into the tail vein of tumor-bearing rats. One hour after intravenous injection, the uptake of 65Zn, 83Rb and 54Mn was relatively high in C6 glioma of 15 radionuclides detected, and was much higher than in other brain regions and in the blood. It is likely that rubidium and manganese, in addition to zinc, are preferentially taken up by tumors in the brain.

Induction of Vasoactive Substances Differs in LPS-Induced and TF-Induced DIC Models in Rats

We have investigated the role of two vasoactive substances, nitric oxide (NO)and endothelin (ET), in the pathophysiology of disseminated intravascular coagulation (DIC), using two types of DIC models. Experimental DIC was induced by sustained infusion of 0.1, 1, 10, or 50 mg/kg lipopolysaccharide (LPS), or 3.75 U/kg thromboplastin (TF), for 4 h via the rat tail vein. Plasma levels of both NOX (metabolites of NO) and ET were significantly increased following infusion of 0.1 mg/kg or greater of LPS in the LPS-induced DIC rat model. In contrast, although a marked increase in the plasma levels of NOX was observed, only a slight increase in plasma ET levels was seen in the TF-induced DIC rat model. No significant differences in the plasma levels of platelets or thrombin-ATIII complex were observed among the TF-induced and LPS (50 mg/dl)-induced DIC models. However, plasma NOX levels rose significantly higher in the TF-induced model, relative to the LPS-induced model (p <0.01). Conversely, plasma ET levels were significantly greater after LPS-induction, compared to TF-induction, of DIC (p <0.01). Vasoconstriction, as well as depressed fibrinolytic activity, may be additional factors leading to severe organ dysfunction in the LPS-induced DIC rat model. Moreover, vasodilatation, as well as enhanced fibrinolytic activity, may help to prevent rats from severe organ dysfunction in the TF-induced DIC model. Our results suggest that modulator of vasoactive substances should be examined in the treatment of DIC.

Safety Evaluation of Lipase Produced from Candida rugosa: Summary of Toxicological Data

The toxicity of lipase AY, an enzyme preparation used in lipid hydrolysis to produce flavors, was evaluated in a series of studies. A 13-week dietary toxicity study in Sprague–Dawley (Crj:CD) rats was conducted in which animals received lipase AY in the feed at concentrations of 0, 625, 1250, or 2500 mg/kg body wt. No adverse treatment-related effects were observed. Lack of genotoxic potential was demonstrated by the results of an in vitro reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and in Escherichia coli strain WP2 uvrA, by an in vitro forward mutation assay in L5178Y mouse lymphoma cells, and by an in vitro chromosome aberration test in CHL/IU cells derived from fibroblasts of the lungs of Chinese hamsters. Finally, the particular strain of Candida rugosa, the yeast strain used to prepare lipase AY, has been shown to be nonpathogenic upon a single injection into the tail vein of rats of viable spores at doses up to 1.5×107 colony-forming units per animal. The results of these studies demonstrate that the enzyme preparation may be considered safe to workers and consumers when employed in the production of flavors from fats.

Intravenous readministration of an adenoviral vector performed long after the initial administration failed to induce re-expression of the original transgene in rats.

Although most humans have been exposed to wild-type adenoviruses in their childhood, titers of neutralizing antibodies against viruses decrease with the passage of time. In the present study, we infused adenoviruses carrying the lacZ gene into the tail vein of rats, and re-infused the same adenoviruses long after the initial administration. However, development of neutralizing antibodies against adenovirus and proliferation of adenovirus-specific T cells were elicited profoundly by adenoviral readministration, and transgene expression was not induced in rats. Our results may have important implications for efficacy considerations when adenoviral vectors are employed in clinical settings for the treatment of cancer.

Safety Evaluation of Phosphodiesterase Produced from Penicillium citrinum: Summary of Toxicological Data

The toxicity of Enzyme RP-1, an enzyme preparation used to hydrolyze yeast RNA to produce flavor enhancers for use in the food industry, was evaluated in a series of studies. A 5-week dietary toxicity study in Wistar rats was conducted in which animals received Enzyme RP-1 in feed at concentrations of 0, 500, 2000, or 8000 mg/kg body wt/day. A 13-week dietary toxicity study in Sprague-Dawley rats was conducted in which animals received RP-1 concentrate at 0, 0.125, 0.5, or 2% in their diets. At the highest dose levels in both studies, submandibular glands in the oral cavity were enlarged, an effect attributed to protease activity of the enzyme preparation. The no-observed-effect level in rats in the 13-week study was 0.5%, equivalent to 317 mg/kg body wt/day for males and 346 mg/kg body wt/day for females. Based on estimated dietary exposure to the enzyme preparation, the margin of exposure is estimated to be greater than 38,000. Lack of genotoxic potential was demonstrated by an in vitro reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and in Escherichia coli strain WP2uvr and by an in vitro chromosome aberration test in CHL/IU cells derived from fibroblasts from the lungs of Chinese hamsters. Finally, the particular strain of Penicillium citrinum, the fungal strain used to prepare Enzyme RP-1, was shown to have low pathogenicity upon a single injection into the tail vein of rats of viable spores at doses up to 2.8×105 colony-forming units per animal. The results of these studies demonstrate that the enzyme preparation may be considered safe to workers and consumers when employed in the production of flavor enhancers from yeast.

Receptor-mediated transfer of DNA--galactosylated poly-L-lysine complexes into mammalian cells in vitro and in vivo.

With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA-poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA--poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA--poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.

Systemic administration of TerplexDNA system: pharmacokinetics and gene expression.

The aim of this study is to extend our previous studies to investigate the TerplexDNA synthetic gene carrier system in pharmacokinetics, biodistribution, and gene expression in major organs after systemic administration. The stability of the TerplexDNA system was analyzed in vitro with a serum incubation assay. The TerplexDNA PK/PD studies were conducted by quantitation of Terplex/radiolabeled DNA [CTP alpha-32P] complexes after rat-tail vein injection. The effect of the TerplexDNA system on gene expression in mouse major organs was analyzed by measuring luciferase activities after systemic administration. The TerplexDNA gene carrier showed significantly longer retention in the vascular space than naked plasmid DNA alone. At early time points (1 h postvenous injection), the lung was the major organ of the TerplexDNA distribution, followed by the liver as a major distribution organ at later time points (24 h postinjection). The major organs of transgene expression after intravenous injection were the liver and heart. The TerplexDNA system has the potential for in vivo applications due to its higher bioavailability of plasmid DNA in the tissues, and due to its organ specific distribution.