CF-1 Mouse Research Articles

In Vitro Evaluation and Biodistribution Studies of HPMA Copolymers Targeting the Gastrin Releasing Peptide Receptor in Prostate Cancer.

The development of diagnostic and therapeutic agents utilizing small peptides (e.g., bombesin (BBN)) to target the overexpression of the gastrin-releasing peptide receptor (GRPR) in cancers has been widely investigated. Herein, we examine the capabilities of BBN-modified HPMA copolymers to target the GRPR. Four positive, four negative, and two zwitterionic BBN HPMA copolymer conjugates of varying peptide content and charge were synthesized. In vitro and in vivo studies were conducted in a GRPR-overexpressing prostate cancer cell line (PC-3) and a normal CF-1 mouse model, respectively. Cellular uptake of the conjugates were found to be charge and BBN density dependent. The positively-charged conjugates illustrated a direct relationship between the extent of cellular internalization, ranging from 0.7 to 20%, and BBN-incorporation density. The negative and zwitterionic conjugates showed low PC-3 uptake values. Blocking studies confirmed the GRPR-targeting effect of the positively-charged constructs. In vivo studies of the positively-charged copolymers resulted in rapid blood clearance by the mononuclear phagocyte system (MPS)-associated tissues (e.g., liver and spleen). Positively-charged BBN-HPMA copolymer conjugates demonstrated good GRPR-targeting and internalization in vitro. However, the impact of peptide density and charge on in vivo MPS recognition are parameters that must be optimized in future agent development.

Propagation of Giardia duodenalis cysts in immunosuppressed CF-1 mice

This study developed and evaluated Giardia duodenalis cyst propagation using a dexamethasone immunosuppressed CF-1 mouse model as an alternative to a previously described Mongolian gerbil model. The CF-1 mouse model shed significantly more cysts per animal during a 16–18 h collection period compared to the gerbil (averages: 7.8 × 106 cysts/CF-1 mouse and 2.5 × 106 cysts/gerbil). In addition, the patency period for this model differed from both G. muris in mice and G. duodenalis in gerbils in that cysts were shed continuously for over 20 days. Results further showed that the β-giardin gene sequences from gerbil derived and mouse derived G. duodenalis were identical, after 34 serial passages through the CF-1 mouse model. Overall, the CF-1 mouse model produced higher concentrations of cysts per animal, and were genetically and phenotypically stable based on β-giardin gene sequences.

Cellular and molecular oxidative stress-related effects in uterine myometrial and trophoblast-decidual tissues after perigestational alcohol intake up to early mouse organogenesis.

The placenta plays a major role in embryo-fetal defects and intrauterine growth retardation after maternal alcohol consumption. Our aims were to determine the oxidative status and cellular and molecular oxidative stress effects on uterine myometrium and trophoblast-decidual tissue following perigestational alcohol intake at early organogenesis. CF-1 female mice were administered with 10% alcohol in drinking water for 17days prior to and up to day 10 of gestation. Control females received ethanol-free water. Treated mice had smaller implantation sites compared to controls (p<0.05), diminished maternal vascular lumen, and irregular/discontinuous endothelium of decidual vessels. The trophoblast giant cell layer was disorganized and presented increased abnormal nuclear frequency. The myometrium of treated females had reduced nitrite content, increased superoxide dismutase activity, and reduced glutathione (GSH) content (p<0.05). However, the trophoblast-decidual tissue of treated females had increased nitrite content (p<0.05), increased GSH level (p<0.001), increased thiobarbituric acid-reactive substance concentration (p<0.001), higher 3-nitrotyrosine immunoreaction, and increased apoptotic index (p<0.05) compared to controls. In summary, perigestational alcohol ingestion at organogenesis induced oxidative stress in the myometrium and trophoblast-decidual tissue, mainly affecting cells and macromolecules of trophoblast and decidual tissues around early organogenesis, in CF-1 mouse, and suggests that oxidative-induced abnormal early placental formation probably leads to risk of prematurity and fetal growth impairment at term.

Oxidative stress and cellular and tissue damage in organogenic outbred mouse embryos after moderate perigestational alcohol intake.

Perigestational alcohol consumption by CF-1 mouse, from before mating up to the period of embryo organogenesis, leads to retarded early embryo development and neural tube defects. Here, we addressed if perigestational alcohol ingestion up to Day 10 of pregnancy induces oxidative stress and changes in macromolecules and organ tissues of early organogenic embryos. Adult CF-1 female mice were administered 10% ethanol in their drinking water for 17 days prior to mating and until Day 10 of gestation, whereas control females were administered ethanol-free water. Our results demonstrated significantly reduced Catalase abundance and activity and increased glutathione content in the embryos of ethanol-treated females. The nitrite level was significantly reduced, but TBARS (thiobarbituric acid reactive substances) content, an index of lipid peroxidation, did not change. Embryos derived from ethanol-treated females also showed higher abundance of 3-nitrotyrosine (3-NT)-containing proteins in all tissues, compared to the control group. Apoptosis was significantly increased in the ectoderm and mesoderm, but not in the heart-although this organ did contain more cleaved Caspase-3-positive cardiomyocytes per area of ventricular myocardium than controls. In sum, moderate perigestational alcohol ingestion up to Day 10 of gestation in mice induces oxidative stress by altering radical nitrogen species and antioxidant enzymatic and non-enzymatic mechanisms in embryos. Further, generalized protein nitration, due to unbalanced nitric oxide levels associated with tissue-specific apoptosis, was detected in embryos, suggesting that oxidative mechanisms may play an important role in the perigestational alcohol-induced malformation of organogenic embryos exposed to ethanol.

Simultaneous Study of Cholesterol and GM1 Ganglioside by Specific Probes: Lipid Distribution during Maturation, Capacitation and the Acrosome Reaction

Lipid distribution has been extensively studied in the plasma membrane of the male gamete due to its relevant role in sperm physiology. However, there are controversial reports about their distribution. Because of different reports about this topic, the aim of this work was define cholesterol and GM1 ganglioside location by simultaneous detection during sperm maturation, capacitation and the acrosome reaction in CF1 mouse sperm. We observed that throughout the epididymis, cholesterol detected by filipin III or complex and GM1 ganglioside by cholera toxin, colocalized in the acrosomal domain of the head and midpiece of the flagellum. During sperm capacitation, a decrease of cholesterol can be observed like diffuse pattern on the whole head, while GM1 ganglioside was restricted to the apical acrosome. Capacitated sperm incubated with acrosome reaction inducers showed a diffuse location on the head with both probes after acrosomal exocytosis. Also, both probes did not show different locations on the flagellum. These findings indicate that cholesterol and GM1 ganglioside have a dynamic behaviour during the life of the sperm, which could indicate that a continuous assembly or disassembly of the lipid raft could be involved in the responsiveness of the sperm to different environments. Thus, during sperm capacitation and acrosome reaction there is a redistribution of the lipids. However, there are not apparent changes during sperm maturation. Cholesterol efflux, a characteristic event of sperm capacitation, could be analyzed through diffuse pattern described by filipin, and GM1 ganglioside patterns observed by the cholera toxin could be interesting when it is needed to evaluate sperm capacitation and the acrosome reaction in fixed sperm.

Ferrozine-Based Iron Quantification of Iron Overloaded Mice: a Simple, Robust Method for the Assessment of Iron Chelation Therapies

Introduction: We describe a low heat nitric acid digestion and colorimetric ferrozine-based iron assay that provides a fast, inexpensive and accurate alternative to high temperature animal tissue processing and inductively coupled plasma mass spectrometry (ICP). This technique is useful for the quantification of iron in iron overloaded animal models and diseases such as β-thalassemia, sickle cell anemia, and myelodysplastic syndromes. We applied it to evaluate iron removal by iron chelation therapies such as liposome-encapsulated deferoxamine (LDFO).Methods: CF-1 mouse liver, spleen, heart, plasma, urine and feces were digested in nitric acid (70%) at 65 °C for 1-2 hours. For large tissues such as the liver, tissues were sectioned at 50 mg fractions (n = 4) to also assess iron homogeneousness. Digested samples were bleached with hydrogen peroxide (30%) and diluted with water before iron quantification by ICP or the ferrozine-based assay. For the ferrozine-based assay, nitric acid was neutralized with ammonium acetate and iron was reduced with ascorbic acid before reaction with ferrozine for the colorimetric assessment of the ferrozine-ferrous iron complex at 550 nm. For iron overloaded models, CF-1 mice (n = 4) were loaded I.V. with iron dextran, iron sucrose, liposome encapsulated iron, or horse ferritin for over a week before sacrifice. Iron removal studies of tissues and excreta from iron overloaded CF-1 (n = 4) started 2 weeks after iron dextran overloading. Animals were dosed with either deferoxamine (DFO) and LDFO by I.V. and Exjade by oral gavage. Urine and feces were collected daily at the start of treatment. Animals were sacrificed 7 days post treatment. Tissues and excrements were digested and measured for iron by the ferrozine-based assay.Results: The ferrozine-based tissue iron quantification assay yields high iron recovery from mouse tissue. CF-1 liver spiked with iron dextran or horse ferritin had an iron recovery of 99±2% and 97±2%, respectively. Liver iron measurements of iron dextran overloaded mouse models resulted in identical iron measurements for the ferrozine-based assay and ICP. For CF-1 mice treated with iron dextran I.V. (0, 100, 300, 600 mg/kg, n = 4), liver iron is highly correlative between the two iron quantification techniques with a slope of 1.03 and R2 of 0.999. In addition, there is a linear iron overloading effect in both the liver (R2 = 0.99) and spleen (R2 = 0.98). CF-1 mice were also iron overloaded with iron sucrose, liposome encapsulated iron, and horse ferritin with dose dependent tissue overload. For all iron overloaded models tested, liver iron and spleen iron were homogenous per tissue when multiple sections were analyzed with an average low 6% difference in iron content. Despite high liver and spleen overloading, none of these iron carriers resulted in statistically significant heart iron overload. Iron dextran overloaded CF-1 mice (100 mg/kg) were treated with LDFO, DFO, and Exjade. LDFO at 100 mg/kg I.V. greatly reduces iron levels in the liver (p = 0.019) and spleen (p = 0.014) compared to non-effective no treatment, free DFO (p = 0.3), and empty liposomes (p = 0.1). Exjade at 30 mg/kg by oral gavage did not result in statistically significant iron removal in the liver or spleen (p < 0.2). Over the first four days, urine and feces were collected daily and also analyzed for iron. Results revealed that iron clearance by LDFO is primarily in the urine (p = 0.022 urine; p = 0.8 feces) while Exjade removed iron appeared in the feces (p = 0.06 feces; p = 0.013 urine). During this short period, drug efficiency in iron excretion (5%) from one dose of the novel LDFO at 100 mg/kg was equivalent to four daily doses of the Exjade at 50 mg/kg/dose.Conclusion: The low heat nitric acid digestion and ferrozine-based tissue iron quantification assay is a simple, precise, highly reproducible tool for the assessment of tissue and excretion iron. The assay enabled the rapid, low cost evaluation of novel iron chelation therapies. We gratefully acknowledge support by NIH SBIR Phase 1 Grant 1R43HD075429-01 and NIH SBIR Phase 2 Grant 2R44HD075429-02. DisclosuresTran:Zoneone Pharma, Inc.: Employment. Petersen:Zoneone Pharma, Inc.: Employment. Noble:Zoneone Pharma, Inc.: Employment, Equity Ownership. Hayes:Zoneone Pharma, Inc.: Employment, Equity Ownership. Working:Zoneone Pharma, Inc.: Consultancy, Equity Ownership. Szoka:Zoneone Pharma, Inc.: Consultancy, Equity Ownership.

Ibuprofen slows migration and inhibits bowel colonization by enteric nervous system precursors in zebrafish, chick and mouse

Hirschsprung Disease (HSCR) is a potentially deadly birth defect characterized by the absence of the enteric nervous system (ENS) in distal bowel. Although HSCR has clear genetic causes, no HSCR-associated mutation is 100% penetrant, suggesting gene–gene and gene-environment interactions determine HSCR occurrence. To test the hypothesis that certain medicines might alter HSCR risk we treated zebrafish with medications commonly used during early human pregnancy and discovered that ibuprofen caused HSCR-like absence of enteric neurons in distal bowel. Using fetal CF-1 mouse gut slice cultures, we found that ibuprofen treated enteric neural crest-derived cells (ENCDC) had reduced migration, fewer lamellipodia and lower levels of active RAC1/CDC42. Additionally, inhibiting ROCK, a RHOA effector and known RAC1 antagonist, reversed ibuprofen effects on migrating mouse ENCDC in culture. Ibuprofen also inhibited colonization of Ret+/− mouse bowel by ENCDC in vivo and dramatically reduced bowel colonization by chick ENCDC in culture. Interestingly, ibuprofen did not affect ENCDC migration until after at least three hours of exposure. Furthermore, mice deficient in Ptgs1 (COX 1) and Ptgs2 (COX 2) had normal bowel colonization by ENCDC and normal ENCDC migration in vitro suggesting COX-independent effects. Consistent with selective and strain specific effects on ENCDC, ibuprofen did not affect migration of gut mesenchymal cells, NIH3T3, or WT C57BL/6 ENCDC, and did not affect dorsal root ganglion cell precursor migration in zebrafish. Thus, ibuprofen inhibits ENCDC migration in vitro and bowel colonization by ENCDC in vivo in zebrafish, mouse and chick, but there are cell type and strain specific responses. These data raise concern that ibuprofen may increase Hirschsprung disease risk in some genetically susceptible children.

Estimation of genetic variability level in inbred CF1 mouse lines selected for body weight.

Estimation of genetic variability level in inbred CF1 mouse lines selected for body weight.

(‐)‐Epigallocatechin 3‐gallate decreases markers of inflammation in a mouse model of ulcerative colitis (1045.24)

Ulcerative colitis (UC) is an inflammatory bowel disease that manifests as constant/cyclic episodes of colonic inflammation, oxidative stress, increased intestinal permeability, and ulceration followed by periods of quiescence. UC increases risk of colon cancer. Green tea polyphenols, such as epigallocatechin‐3‐gallate (EGCG), have been shown to inhibit oxidative stress in animal and cell studies. Here, we examined the anti‐inflammatory effects of EGCG in the dextran sulfate sodium (DSS)‐induced CF‐1 mouse model of UC. Following induction of colitis by treatment with 1.5% DSS for 7 days, mice were treated with EGCG (0.32%) as the sole source of drinking fluid for 3 days. In the colon homogenate, inflammatory cytokine levels of IL‐6 and TNF‐α were decreased by 50% and IL‐1β by 25% as a result of the EGCG administration. Plasma inflammatory markers and endotoxin levels were also decreased. These results suggest that EGCG can decrease both inflammatory response and intestinal permeability in the DSS‐induced UC mouse model. Ongoing mechanistic studies are focused on determining the effect of EGCG on gut barrier function.

Matrix metalloproteinase expression and activity in trophoblast-decidual tissues at organogenesis in CF-1 mouse

During early placentation, matrix metalloproteinases (MMPs) play important roles in decidualization, trophoblast migration, invasion, angiogenesis, vascularization and extracellular matrix (ECM) remodeling of the endometrium. The aim of our study was to analyze the localization, distribution and differential expression of MMP-2 and -9 in the organogenic implantation site and to evaluate in vivo and in vitro decidual MMP-2 and -9 activities on day 10 of gestation in CF-1 mouse. Whole extracts for Western blotting of organogenic E10-decidua expressed MMP-2 and -9 isoforms. MMP-2 immunoreactivity was found in a granular and discrete pattern in ECM of mesometrial decidua (MD) near maternal blood vessels and slightly in non-decidualized endometrium (NDE). Immunoexpression of MMP-9 was also detected in NDE, in cytoplasm of decidual cells and ECM of vascular MD, in trophoblastic area and in growing antimesometrial deciduum. Gelatin zymography showed that MMP-9 activity was significantly lower in CM compared to the active form of direct (not cultured) and cultured decidua. The decidual active MMP-9 was significantly higher than the active MMP-2. These results show differential localization, protein expression and enzymatic activation of MMPs, suggesting specific roles for MMP-2 and MMP-9 in decidual and trophoblast tissues related to organogenic ECM remodeling and vascularization during early establishment of mouse placentation.

Quantitative and semiquantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells

Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule, sICAM (ICAM-1, CD54), was expressed by CF-1 cells, but not STO cells, and similarly, the cell matrix-associated molecules endocan (endothelial cell-specific molecule 1), endostatin (collagen XVIII), and matrix metalloproteinase 3 were expressed more by CF-1 cells. Tissue inhibitor of metalloproteinases 1 was robustly expressed by both feeder cells. Other proteins primarily detected from CF-1 cells included retinol-binding protein 4 and FGF21, while STO cells secreted more interferon gamma. Both feeder cells produced no or low amounts of LIF, tumor necrosis factor alpha, vascular endothelial growth factor (VEGF), VEGF-B, prolactin, various interleukins, fibroblast growth factor (FGF)-1, FGF-2, FGF-7, EGF, HB-EGF, and amphiregulin. The results may explain some of the cell growth and maintenance responses by various types of cells co-cultured on STO or CF-1 feeder cells.

Nucleolar organizer regions in a chronic stress and oral cancer model

This study aimed to examine the role of chronic restraint stress (RS) on oral squamous cell carcinomas induced by 4-nitroquinoline-1-oxide (4-NQO) in CF-1 mouse tongues, measured by the expression of argyrophilic staining of nucleolar organizer regions (AgNOR). Thirty one samples of lingual epithelial tissue of CF-1 mice with a diagnosis of oral squamous cell carcinoma (OSSC) were assigned to two experimental groups: the RS/4-NQO group, where animals received RS and induction of oral chemical carcinogenesis (n=17); and the 4-NQO group, where animals received induction of chemical carcinogenesis without restraint stress (n=14). The mean number and distribution pattern of AgNOR were recorded. The mean AgNOR number per cell was found to be slightly higher in the 4-NQO group. AgNOR in the RS/4-NQO group revealed a higher tendency to be arranged in a clumped distribution compared to the 4-NQO group. No statistically significant difference was found between the groups. In conclusion, the induction of chronic restraint stress in CF-1 mice does not increase the number or affect the distribution pattern of AgNORs in OSSC induced by 4-NQO.

Embryo developmental disruption during organogenesis produced by CF‐1 murine periconceptional alcohol consumption

The aim was to study the control females (CF)-1 mouse embryo differentiation, growth, morphology on embryonic E- and N-cadherin expression at midgestation after periconceptional moderate alcohol ingestion. Adult female mice were exposed to 10% ethanol in drinking water for 17 days previous to and up to day 10 of gestation (ethanol-exposed females, EF) and were compared with nonexposed CF. EF presented reduced quantities of E10 to E10.5 embryos, greater percentage of embryos at stages less than E7.5, reduced implantation site numbers/female, and increased resorptions compared with CF. EF-embryo growth was significantly affected as evidenced by reduced cephalic and body sizes of E10 and E10.5 embryos (scanning electron microscopy) and decreased protein content of E10.5 embryos vs. CF embryos. A significantly higher percentage of EF-E10-10.5 embryos presented abnormal neural tube (NT) closure vs. the percentage of CF. E10 embryos from EF presented elevated tissue disorganization, pyknosis and nuclear condensation in somites, mesenchymal and neuroepithelial tissue. Immunohistochemical E- and N-cadherin distribution patterns were similar in organic structures of E10 embryos between groups. However, western blot revealed that E- and N-cadherin expression levels were significantly increased in EF-derived embryos vs. controls. Perigestational ethanol consumption by CF-1 mice induced significant damage in the organogenic embryogenesis by producing delayed differentiation, growth deficiencies, and increasing the frequency of NT defects. Ethanol exposure may disrupt cell-cell adhesion leading to upregulation of E- and N-cadherin expression suggesting that deregulation of cell adhesion molecules could be involved in the disruption of embryo development at organogenesis in CF-1 mouse.

Mouse Embryos Stressed by Physiological Levels of Osmolarity Become Arrested in the Late 2-Cell Stage Before Entry into M Phase1

Preimplantation mouse embryos of many strains become arrested at the 2-cell stage if the osmolarity of culture medium that normally supports development to blastocysts is raised to approximately that of their normal physiological environment in the oviduct. Arrest can be prevented if molecules that serve as "organic osmolytes" are present in the medium, because organic osmolytes, principally glycine, are accumulated by embryos to provide intracellular osmotic support and regulate cell volume. Medium with an osmolarity of 310 mOsM induced arrest of approximately 80% of CF1 mouse embryos at the 2-cell stage, in contrast to the approximately 100% that progressed beyond the 2-cell stage at 250 or 301 mOsM with glycine. The nature of this arrest induced by physiological levels of osmolarity is unknown. Arrest was reversible by transfer to lower-osmolarity medium at any point during the 2-cell stage, but not after embryos would normally have progressed to the 4-cell stage. Cessation of development likely was not due to apoptosis, as shown by lack of external annexin V binding, detectable cytochrome c release from mitochondria, or nuclear DNA fragmentation. Two-cell embryos cultured at 310 mOsM progressed through the S phase, and zygotic genome activation markers were expressed. However, most embryos failed to initiate the M phase, as evidenced by intact nuclei with decondensed chromosomes, low M-phase promoting factor activity, and an inactive form of CDK1, although a few blastomeres were arrested in metaphase. Thus, embryos become arrested late in the G(2) stage of the second embryonic cell cycle when stressed by physiological osmolarity in the absence of organic osmolytes.

Effect of pregnane X receptor (PXR) prototype agonists on chemoprotective and drug metabolizing enzymes in mice

The effects of known PXR inducers; spironolactone [SPL, (i.p.)], pregnenolone-16 alpha-carbonitrile [PCN, (i.g.)] and dexamethasone (i.p.) on hepatic drug metabolizing enzymes in the male CF1 mouse were examined 24h after 3 daily doses (50, 100, or 200mg/kg) in corn oil vehicle. All three compounds produced dose-dependent elevations in cytochrome P450 [CYP3A], glutathione S-transferase [GST] and NAD(P)H quinone oxidoreductase [NQO] activities. Only elevations in CYP3A produced after dexamethasone were statistically significant. An elevation in microsomal epoxide hydrolase [mEH, Ephx1] activity was seen after almost all treatments but was erratic with dose. UDP-glucuronosyltransferase and thioredoxin reductase activities were not increased by any agent. Dexamethasone elevated Cyp1a1/2 mRNA at the low dose but reduced the mRNA transcript and activity of the enzyme at the mid and high doses. The mRNA responses of Ephx1 and Nqo1 showed a close parallel to each other with no increases after dexamethasone or SPL treatment, and elevations at the mid dose of PCN. With the exception of dexamethasone at the high dose, elevations in Gst mRNAs were seen with all doses of the three agents. When a large number of hepatic enzymes are examined, the responses to dexamethasone, SPL and PCN are far from identical, and any dose dependency is agent specific. Induction of enzymes seems more complicated to be controlled by one orphan receptor. This study not only filled the void about the murine PXR-induction profile but also will help in the course of drug development research with respect to extrapolation to human risk assessment.

Labeling, stability and biodistribution studies of 99mTc-cyclized Tyr3-octreotate derivatives

To probe the interplay between radiotracer stability and somatostatin receptor affinity, Tyr(3)-octreotate and six variations of its peptide sequence, for which the Re-cyclized products were previously reported, were radiolabeled with (99m)Tc and investigated for their in vitro stability. Radiolabeling of the peptides was effected by ligand exchange from (99m)Tc-glucoheptonate, and the desired products were purified by radio-RP-HPLC. The in vitro stability in phosphate buffered saline, mouse serum and cysteine solutions at physiological temperature and pH for all seven (99m)Tc-cyclized peptides was determined by radio-RP-HPLC and radio-TLC. Normal CF-1 mouse biodistribution studies were performed for three of the (99m)Tc-cyclized peptides. Based on the fully characterized Re-cyclized peptide analogues, four (99m)Tc-coordination motifs were proposed for the (99m)Tc-cyclized peptides. Technetium-99m-cyclized Tyr(3)-octreotate derivatives with N(2)S(2) metal coordination modes and large metal ring sizes were susceptible to oxidation and loss of (99m)Tc in the form of (99m)TcO(4)(-), as evidenced by their instability in the various solutions under physiological conditions (15-58% intact at 24 h). As anticipated, the addition of a third cysteine to the sequence stabilized the (99m)Tc metal coordination, and peptides with NS(3) coordination modes remained >85% intact out to 24 h. No significant differences were observed in the biodistribution studies performed with three peptides of varying stabilities. Improvements in stability were not sufficient to outweigh the low somatostatin receptor affinity for the peptides in this study. Further improvements in the peptide sequence and/or metal coordination are needed to result in a radiodiagnostic/radiotherapeutic pair for targeting the somatostatin receptor.

Transgenerational developmental effects and genomic instability after X-irradiation of preimplantation embryos: Studies on two mouse strains

Recent results have shown that irradiation of a single cell, the zygote or 1-cell embryo of various mouse strains, could lead to congenital anomalies in the fetuses. In the Heiligenberger strain, a link between the radiation-induced congenital anomalies and the development of a genomic instability was also suggested. Moreover, further studies showed that in that strain, both congenital anomalies and genomic instability could be transmitted to the next generation. The aim of the experiments described in this paper was to investigate whether such non-targeted transgenerational effects could also be observed in two other radiosensitive mouse strains (CF1 and ICR), using lower radiation doses.Irradiation of the CF1 and ICR female zygotes with 0.2 or 0.4Gy did not result in a decrease of their fertility after birth, when they had reached sexual maturity. Moreover, females of both strains that had been X-irradiated with 0.2Gy exhibited higher rates of pregnancy, less resorptions and more living fetuses. Additionally, the mean weight of living fetuses in these groups had significantly increased.Exencephaly and dwarfism were observed in CF1 fetuses issued from control and X-irradiated females. In the control group of that strain, polydactyly and limb deformity were also found. The yields of abnormal fetuses did not differ significantly between the control and X-irradiated groups. Polydactyly, exencephaly and dwarfism were observed in fetuses issued from ICR control females. In addition to these anomalies, gastroschisis, curly tail and open eye were observed at low frequencies in ICR fetuses issued from X-irradiated females. Again, the frequencies of abnormal fetuses found in the different groups did not differ significantly.In both CF1 and ICR mouse strains, irradiation of female zygotes did not result in the development of a genomic instability in the next generation embryos.Overall, our results suggest that, at the moderate doses used, developmental defects observed after X-irradiation of female zygotes of these two sensitive mouse strains should not be transmitted to the next generation. Paradoxically, other studies would be needed to address the question of a potential increase of fertility after doses lower than 0.2Gy in both strains.

The effect of superovulation on the contributions of individual blastomeres from 2-cell stage CF1 mouse embryos to the blastocyst

It remains controversial whether blastomeres of 2-cell stage mouse embryos show bias in their contribution to the blastocyst and whether there is any effect of superovulation. Two-cell stage embryos from CF1 mice were derived by either natural breeding (N) or superovulation (S) and cultured in vitro. At blastocyst, inner cell mass and trophectoderm were distinguished by Cdx2 and Oct4 immunostaining. A fluorescent dye (CM-Dil) was also used to tag individual blastomeres at the 2-cell stage, and the descendant cells identified by their red fluorescence. S and N embryos developed to blastocyst at the same rate and contained a similar number of cells. However, with S embryos, the descendants of the blastomere labeled with CM-DiI contributed predominantly to either the embryonic or abembryonic pole about 70% of the time, whereas most N embryos displayed random patterning, with no restriction to one or other of the poles. In S-embryos, but not N-embryos, the leading blastomere at second cleavage contributed preferentially to the embryonic pole of the blastocyst and the lagging blastomere to the abembryonic pole and hence mural trophectoderm. In addition, a tetrahedral rather than a flat morphology at the 4-cell stage of S-embryos was strongly biased to displaying the embryonic/abembryonic pattern at blastocyst. In contrast, S-embryos lacking a zona pellucida resembled N embryos in their patterning. In CF1 mice, superovulation has little effect on development to blastocyst, but enforces a greater degree of lineage restriction than natural breeding, most likely through constraints imposed by the zona pellucida.

MDR1A (ABCB1)-Deficient CF-1 Mutant Mice are Susceptible to Cerebral Malaria Induced by Plasmodium Berghei ANKA

Under experimental conditions, Plasmodium berghei infection causes cerebral malaria (CM) in susceptible strains of mice such as C57BL/6 and CBA/Ca, whereas BALB/c or DBA/2J strains serve as a model for CM-resistant mice. The aim of the present study was to investigate the susceptibility of the CF1 mouse strain, carrying a spontaneous mutation of the mdr1a gene, to infection with Plasmodium berghei ANKA (PbA). The mdr1a gene codes for P-glycoprotein (P-gp/ABCB1), an efflux pump that is one of the major components of the blood-brain barrier. P-gp effluxes a broad range of xenobiotics from the brain to blood, preventing accumulation and toxicity in the central nervous system. CFI mdr1a (-/-) mice are used to investigate drug transport by efflux pumps. Because many antimalarial agents are effluxed by P-gp (mefloquine, quinine), it was important to determine whether CF1 mice can develop cerebral malaria to predict drug toxicity during cerebral malaria. Our work showed that CF1 mdr1a (-/-) mice are susceptible to PbA. CF1 and C57BL/6N mice (the reference strain) infected with PbA have similar profiles with regard to clinical signs, brain histological lesions, and brain macrophagic activation observed by immunohistological methods.

In vitro culture under physiologic oxygen concentration improves blastocyst yield and quality: a prospective randomized survey on sibling oocytes

A prospective randomized study was carried on sibling oocytes in which human gametes and embryos were cultured at physiologic (6% CO(2) + 5% O(2) + 89% N(2)) condition from insemination to day 6 or at atmospheric (6% CO(2) in air) condition until day 3 and at physiologic environment afterward. The results showed that physiologic oxygen concentration throughout culture period improved total blastocyst yield and day 5 embryo quality.