Fischer F344 Rats Research Articles

Restrictive model of compensated carbon tetrachloride-induced cirrhosis in rats

To develop a simplified and quick protocol to induce cirrhosis and standardize models of partial liver resection in rats. In Fischer F344 rats two modified protocols of phenobarbital-carbon tetrachloride (CCl4) (dilution 50%) gavage to induce cirrhosis (frequency adjusted according to weight, but each subsequent dose was systematically administered) were tested, i.e. the rapid and slow protocols. Prothrombin time (PT) and total bilirubin (TB) were also evaluated. Animals from the rapid group underwent 15% hepatectomy and animals from the slow group underwent 70% hepatectomy. Rapid protocol: This corresponded to 1 gavage/4 d over 6 wk (mortality 30%). Mean PT was 35.2+/-2.8 s (normal: 14.5 s), and mean TB was 1.8+/-0.2 mg/dL (normal: 0.1 mg/dL). Slow protocol: This corresponded to 1 gavage/6 d over 9 wk (mortality 10%). Mean PT was 11.8+/-0.2 s (normal: 14.5 s), and mean TB was 0.4+/-0.04 mg/dL (normal: 0.1 mg/dL). Pathological analyses were performed in both protocols which showed persistent cirrhosis at 3 mo. Rat mortality in the rapid gavage group who underwent 15% hepatectomy and in the slow gavage group who underwent 70% hepatectomy was 50% and 70%, respectively. Our modified model is a simplified method to induce cirrhosis which is rapid (6 to 9 wk), efficient and stable up to 3 mo. Using this method, "Child Pugh A" or "Child Pugh BC" cirrhotic rats were obtained. Our models of cirrhosis and hepatectomy can be used in various situations focusing on postoperative survival.

An atlas-based image registration method for dopamine receptor imaging with PET in rats

For analysis of in vivo dopamine receptor binding in the rat brain by positron emission tomography (PET), a convenient method to obtain precise anatomical registration for striatum and cerebellum on the PET image was developed. On the PET measurements, a control, an anesthetized rat was positioned in a stereotaxic holder so that the horizontal plane of the PET image would be parallel to the horizontal plane of the brain atlas. After the positioning, [11C]raclopride was intravenously injected into the rats and scanned to obtain PET images of dopamine D2 receptor in the brain. The striatum was bilaterally identified in the obtained PET image. The atlas-based regions of interest (ROIs) of the whole brain were preliminarily created according to the atlas, and were superimposed on an early phase PET image. The early phase PET image was compatible to the whole brain ROI in the atlas, which enabled determination of striatal and cerebellar ROI difficult to determine by the PET image alone. Using the cerebellar radioactivity as a reference input function, rate constants between the free/nonspecific compartment and the receptor bound compartment (k3 and k4) were calculated by a two-parameter compartment model, and the binding potential (k3/k4) was estimated. The binding potential and its coefficients of variation were 1.56+/-0.30, 19.3% in Wistar rats, 1.05+/-0.14, 13.4% in Sprague-Dawley (SD) rats, and 1.29+/-0.07, 5.2% in Fischer F344 rats, in which binding potential in Wistar rats was significantly higher than that in SD rats. This method is objective and convenient in routine use for PET studies in rats, regardless of differences in the rat strains.

Intravesical Ultrasonography for Tumor Staging in an Orthotopically Implanted Rat Model of Bladder Cancer

Noninvasive and serial evaluation of tumor development and growth is important in an orthotopic animal bladder tumor model. However, to our knowledge a reliable method has not been established. We determined the usefulness of intravesical ultrasonography for tumor staging and volumetric assessment in an animal model. Tumors with various stages were formed in 20 female Fischer F344 rats by implanting AY-27 rat bladder carcinoma cells. Cells were implanted by instilling a suspension (4x10 cells) into the bladder cavity after urothelial denudation using hydrochloric acid or by directly injecting the cell suspension into the bladder wall. Tumor volume and invasion depth were measured by intravesical ultrasonography using an ultrathin 2.5Fr intraluminal ultrasound catheter via the urethra 7 to 10 days after cancer cell implantation. The rats were then sacrificed for histopathological examination. All rats showed bladder tumors 7 to 10 days after cancer cell implantation, of which stages varied from superficial to advanced disease. Intravesical ultrasonography showed a clear cross-sectional view of all layers of the bladder wall and enabled visualization of bladder tumors (minimal 0.5 mm in diameter). This approach also provided an accurate diagnosis of tumor invasion into muscle layers and perivesical tissues with precise invasion depth and tumor size. The positive predictive ratio regarding tumor staging reached 85%. Repeat examinations were feasible without noticeable adverse effects. Intravesical ultrasonography is a reliable and appropriate noninvasive method for evaluating tumor stage and size in an orthotopic rat bladder tumor model.

A ninety-day feeding, subchronic toxicity study of oligo- N-acetylglucosamine in Fischer 344 rats

Oligo- N-acetylglucosamine (OAG) is a hydrolyzed derivative of chitin that has been used as a sweetener in foods. Since, no information has been published about the safety of OAG, a 90-day feeding study was conducted, using F344 Fischer rats of both sexes, to characterize and evaluate the toxicity of OAG, and the results of the study are presented here. Dietary levels of 0% (control), 0.2%, 1%, and 5% OAG did not change any measurements in ophthalmological examinations, clinical signs, body weights, food consumption, hematology, blood biochemistry, urinalysis, necropsies, organ weights or histological examinations. The sole finding, which could not be clarified to be attributed to OAG or not, was a decrease in the relative weight of the submaxillary gland to body weight in the male animals given the 5% OAG diet. Although no lesions were found in either gross or histological examination in the present study, further studies using OAG levels higher than 5% might provide a clue to the mechanisms underlying the decreased organ weight observed here. Taken together, under the conditions in the present study, the No Observed Adverse Effect Level (NOAEL) for males was found to be 1% (0.641 g/kg/day); and that for females, 5% (3.64 g/kg/day) or more, based on the lack of toxicological effects.

Prolonged Amelioration of Acute Lung Allograft Rejection by Overexpression of Human Interleukin-10 Under Control of a Long Acting Ubiquitin C Promoter in Rats

The prolonged effect of electroporation-mediated human interleukin-10 (hIL-10) overexpression in skeletal muscle under the control of the constitutional polyubiquitin C promoter (pUb hIL-10) on rat lung allograft rejection was evaluated. Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Either 2.5 mug pCIK hIL-10 (hIL-10/cytomegalovirus early promoter enhancer) alone (Group I/sacrifice Day 5 and II/sacrifice Day 10) or in combination with 2.5 mug pUb hIL-10 (hIL-10/UbC promoter; Group III/sacrifice Day 10) were injected into the tibialis anterior muscle of the recipient, followed by electroporation 24 hours before transplantation. Animals in Control Groups IV and V without gene transfer were euthanized on Day 5 and 10, respectively. All animals received a daily non-therapeutic dose of cyclosporine A (2.5 mg/kg). In Control Group IV, complete rejection (median A3B3) was noted on Day 5 with a Pao(2) of 43 +/- 9 mm Hg. In recipients of Control Group V, measurement of gas exchange on Day 10 and rejection grading was impossible because of complete destruction of the allograft. Group I animals on Day 5 (233 +/- 123 mm Hg; p = 0.02 vs Group IV) and Group II animals on Day 10 (150 +/- 139 mm Hg; p = 0.15 vs Group IV) demonstrated improved graft function. Graft function in Group III was further improved on Day 10 (299 +/- 123 mm Hg; p = 0.002 vs Group IV; p = 0.05 vs Group II; p = 0.36 vs Group I). Rejection was significantly reduced in Group III (median, A2B2) compared with Group II (median, A4B3; p < 0.05). Interleukin-10 overexpression under control of the constitutive ubiquitin C promoter ameliorates acute rejection and preserves lung graft function for a prolonged time.

Diabetes-Induced Changes in the Alternative Splicing of the Slo Gene in Corporal Tissue

ObjectivesErectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel α-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. MethodsReverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. ResultsA novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. ConclusionsAlternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity.

Chimerism in Lymphoid Tissues and Donor-Specific Antibody Response After Injection of Allogenic Splenic Dendritic Cells From Fischer Rats to Lewis Recipients

The aim of this work was to study cellular chimerism achieved in lymphoid tissues and production of antidonor lymphocyte antibodies after injection of splenic dendritic cells (DCs) from Fischer F344 rats to Lewis recipients, a model of chronic rejection. DCs isolated from the spleen expressed OX62 (95%), CD80 (70%), and CD86 (80%). Two doses of these nonplasmacytoid splenic DCs from Fischer rats (2 × 10 6 and 5 × 10 6), which had been labeled ex vivo with a TRITC fluorochrome (PKH26), were injected to Lewis recipients. Using fluorescence microscopy TRITC positive cells were localized at day 15 and day 45 in frozen sections from spleen, thymus, mesenteric lymph nodes, heart, liver, kidney, and skin ( n = 5 per group). Donor-specific antibodies were sought with flow cytometric crossmatches in serum samples taken at 7, 15, 30, and 45 days. TRITC-positive DCs were essentially localized in the spleen, the thymus, and lymph nodes of Lewis recipients. The majority of DCs were detected in the spleen (14.9 ± 3.3 and 14.3 ± 0.9 DCs/per high power field respectively at day 45). A significant number of DCs was also detected in the thymus and mesenteric lymph nodes at both times. Only some scattered TRITC-positive cells were observed in other organs. The number of DCs was stable over time and did not depend on the injected dose. A positive flow cytometric cross-match was observed at day 30 in all recipients independent of the injected dose. These data showed that 2 × 10 6 mature, nonplasmocytoid DCs from F344 rats injected to Lewis recipients induced stable chimerism in primary and secondary lymphoid organs and a humoral response to donor antigens.

Inflammation and Atrophy Precede Prostatic Neoplasia in a PhIP-Induced Rat Model

2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) has been implicated as a major mutagenic heterocyclicamine in the human diet and is carcinogenic in the rat prostate. To validate PhIP-induced rat prostatic neoplasia as a model of human prostate cancer progression, we sought to study the earliest histologic and morphologic changes in the prostate and to follow progressive changes over time. We fed sixty-seven 5-week-old male Fischer F344 rats with PhIP (400 ppm) or control diets for 20 weeks, and then sacrificed animals for histomorphologic examination at the ages of 25, 45, and 65 weeks. Animals treated with PhIP showed significantly more inflammation (P = .002, > .001, and .016 for 25, 45, and 65 weeks, respectively) and atrophy (P = .003, > .001, and .006 for 25, 45, and 65 weeks, respectively) in their prostate glands relative to controls. Prostatic intraepithelial neoplasia (PIN) occurred only in PhIP-treated rats. PIN lesions arose in areas of glandular atrophy, most often in the ventral prostate. Atypical cells in areas of atrophy show loss of glutathione S-transferase π immunostaining preceding the development of PIN.None of the animals in this study developed invasive carcinomas, differing from those in previous reports. Overall, these findings suggest that the pathogenesis of prostatic neoplasia in the PhIP-treated rat prostate proceeds from inflammation to postinflammatory proliferative atrophy to PIN.

Tumor viability evaluation by positron emission tomography with [18F]FDG in the liver metastasis rat model

We prepared a liver metastatic tumor model by injection of rat colon adenocarcinoma cells to Fischer F344 rats through portal vein, and applied positron emission tomography (PET) using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) ([18F]FDG-PET) to this model. At an early stage of the model, multiple small tumor nodules appeared in the inferior lobes of the livers, and extended later into the superior lobes. To evaluate the tumor growth and tumor viability at the early stage, we proposed a new concept, tumor viability index (TVI), instead of the standardized uptake value (SUV) of the [18F]FDG uptake. The TVI was defined by subtracting the signal based on the normal liver from the total signal in the whole liver including tumor nodules: (whole liver SUV-normal liver SUV) x ml of whole liver region of interest (ROI). For the signal of the whole liver, ROIs were placed on six slices covering the whole liver, and the ROI of normal liver region was located in the superior lobe of the liver. The average TVI values increased with tumor growth and significantly correlated with the numbers of tumor nodules. The new concept may be useful for evaluating the tumor viability non-invasively and quantitatively by [18F]FDG-PET.

Cisplatin Inhibition of Anthrax Lethal Toxin

Bacillus anthracis lethal toxin (LT) produces symptoms of anthrax in mice and induces rapid lysis of macrophages derived from certain inbred strains. LT is comprised of a receptor binding component, protective antigen (PA), which delivers the enzymatic component, lethal factor (LF), into cells. We found that mouse macrophages were protected from toxin by the antitumor drug cis-diammineplatinum (II) dichloride (cisplatin). Cisplatin was shown to inhibit LT-mediated cleavage of cellular mitogen-activated protein kinases (MEKs) without inhibiting LF's in vitro proteolytic activity. Cisplatin-treated PA lost 100% of its ability to function in toxicity assays when paired with untreated LF, despite maintaining the ability to bind to cells. Cisplatin-treated PA was unable to form heptameric oligomers required for LF binding and translocation. The drug was shown to modify PA in a reversible noncovalent manner. Not surprisingly, cisplatin also blocked the actions of anthrax edema toxin and of LF-Pseudomonas aeruginosa exotoxin A fusion peptide (FP59), both of which require PA for translocation. Treatment of BALB/cJ mice or Fischer F344 rats with cisplatin at biologically relevant concentrations completely protected the animals from a coadministered lethal dose of LT. However, treatment with cisplatin 2 hours before or after animals received a lethal bolus of toxin did not protect them.

In vivo observation of pulmonary micrometastasis less than 300 μm in diameter

Pulmonary micrometastases less than 300 μm in diameter could be observed in vivo. The shape of metastases was usually round but some were linear. We defined each type of metastases by the ratio of long axis and short axis of metastasis mass, i.e. round type (R type) is less than three and linear type (L type) is more than three. RCN9 rat colon cancer cells (1x107) labeled with green fluorescent protein (GFP) transfected by calcium-phosphate DNA co-precipitates were transplanted (tp) into the liver of Fischer F344 rats. Three, 4, 6, 8 and 10 weeks after tp, pulmonary micrometastases were observed using a real-time confocal laser scanning microscope in vivo then rats were sacrificed for histology. Blood flow was visualized by intra venous injection of fluorescein isothiocyanate (FITC) labeled red blood cells (RBCs). To visualize vascular endothelium rhodamin 6G or acridine orange was infused just before observation. Size (μm; range of long axis/range of short axis) at each period (w; weeks) after tp are summarized below. Data at 4 and 8 weeks are not shown. Time (n) 3w (5) 6w (7) 10w (3) R type 10~140/10~50 10~300/10~300 10~100/10~80 L type 160/30 32~120/8~30 80~50/15 Size of metastases was not correlated with weeks after tp. This suggests that new metastasis is continuously occurring and some metastases might be cleared. By FITC-RBCs’ movement and the depth of visualized endothelium, we tried to confirm in vivo whether each type of metastasis was inside the pulmonary microvessel or outside.

Identification of novel transcriptional networks in response to treatment with the anticarcinogen 3H-1,2-dithiole-3-thione

3H-1,2-dithiole-3-thione (D3T), an inducer of antioxidant and phase 2 genes, is known to enhance the detoxification of environmental carcinogens, prevent neoplasia, and elicit other protective effects. However, a comprehensive view of the regulatory pathways induced by this compound has not yet been elaborated. Fischer F344 rats were gavaged daily for 5 days with vehicle or D3T (0.3 mmol/kg). The global changes of gene expression in liver were measured with Affymetrix RG-U34A chips. With the use of functional class scoring, a semi-supervised method exploring both the expression pattern and the functional annotation of the genes, the Gene Ontology classes were ranked according to the significance of the impact of D3T treatment. Two unexpected functional classes were identified for the D3T treatment, cytosolic ribosome constituents with 90% of those genes increased, and cholesterol biosynthesis with 91% of the genes repressed. In another novel approach, the differentially expressed genes were evaluated by the Ingenuity computational pathway analysis tool to identify specific regulatory networks and canonical pathways responsive to D3T treatment. In addition to the known glutathione metabolism pathway (P = 0.0011), several other significant pathways were also revealed, including antigen presentation (P = 0.000476), androgen/estrogen biosynthesis (P = 0.000551), fatty acid (P = 0.000216), and tryptophan metabolism (P = 0.000331) pathways. These findings showed a profound impact of D3T on lipid metabolism and anti-inflammatory/immune-suppressive response, indicating a broader cytoprotective effect of this compound than previously expected.

Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design.

Bacillus anthracis secretes three polypeptides: protective antigen (PA), lethal factor (LF), and edema factor (EF), which interact at the surface of mammalian cells to form toxic complexes. LF and EF are enzymes that target substrates within the cytosol; PA provides a heptameric pore to facilitate LF and EF transport into the cytosol. Other than administration of antibiotics shortly after exposure, there is currently no approved effective treatment for inhalational anthrax. Here we demonstrate an approach to disabling the toxin: high-affinity blockage of the PA pore by a rationally designed low-molecular weight compound that prevents LF and EF entry into cells. Guided by the sevenfold symmetry and predominantly negative charge of the PA pore, we synthesized small cyclic molecules of sevenfold symmetry, beta-cyclodextrins chemically modified to add seven positive charges. By channel reconstitution and high-resolution conductance recording, we show that per-6-(3-aminopropylthio)-beta-cyclodextrin interacts strongly with the PA pore lumen, blocking PA-induced transport at subnanomolar concentrations (in 0.1 M KCl). The compound protected RAW 264.7 mouse macrophages from cytotoxicity of anthrax lethal toxin (= PA + LF). More importantly, it completely protected the highly susceptible Fischer F344 rats from lethal toxin. We anticipate that this approach will serve as the basis for a structure-directed drug discovery program to find new and effective treatments for anthrax.

Acute and subacute effects of tobacco alkaloids, tobacco-specific nitrosamines and phenethyl isothiocyanate on N′-nitrosonornicotine metabolism in rats

N′-Nitrosonornicotine (NNN) was the first tobacco-specific nitrosamine (TSNA) identified as carcinogen in tobacco smoke, but no data exist on in vivo interactions between NNN and other tobacco alkaloids, TSNA or phenethyl isothiocyanate (PEITC) which have been demonstrated in various studies on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Acute effects on NNN metabolism were tested in male Fischer F344 rats injected s.c. with 30 nmol/kg body weight (bw) [5- 3H]NNN either alone or simultaneously with 15 μmol/kg bw nicotine, nornicotine, anatabine, or anabasine, 150 μmol/kg bw cotinine, 3 μmol/kg bw myosmine, or 300 nmol/kg bw of either N′-nitrosoanatabine or N′-nitrosoanabasine. Another group of rats was fed a diet supplemented with PEITC at 1 μmol/g diet starting 24 h before NNN treatment. Within 24 h more than 80% and about 10% of the radioactivity was excreted with urine and feces, respectively. Urinary metabolites were separated by reversed-phase radio-HPLC and identified by co-chromatography with UV standards. In two sets of experiments with control rats treated with NNN only, 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid, 44.4/44.8%), 4-oxo-4-(3-pyridyl)butanoic acid (keto acid, 32.4/31.5%), NNN- N-oxide (5.0/3.8%), 4-(3-pyridyl)butane-1,4-diol (diol, 1.1/1.0%) and norcotinine (2.3/1.0%) were consistently detected besides unmetabolised NNN (4.7/3.3%). Co-treatment with nicotine, cotinine, nornicotine and PEITC shifted the contribution of the two major metabolites significantly in favor of hydroxy acid (108–113% of control) as compared to keto acid (86–90% of control). The same treatments also increased norcotinine (135–170% of control). These changes are consistent with a decreased metabolic activation of NNN. In subacute studies rats received NNN in drinking water for 4 weeks at a daily dose of 30 nmol/kg bw with or without nornicotine at 15 μmol/kg bw or myosmine at 3 μmol/kg bw. On the last day of the experiment all rats received [5- 3H]NNN at 30 nmol/kg bw with a contaminated apple bite followed by collection of urine and feces for 18 h. Most of the radioactivity, 87–96% of the dose, was recovered in urine and only minor amounts have been excreted in feces or persisted in blood. In urine of the NNN-control group keto acid (32.2%) and unmetabolised NNN (3.9%) were present in identical amounts as in the acute experiment whereas hydroxy acid (41.4% of total radioactivity in urine, 93% of acute NNN control) was reduced in expense of the minor NNN metabolites. Co-administration of nornicotine resulted in a small but significant rise of keto acid (107% of control) and a significant decrease in NNN- N-oxide (76% of control). After co-treatment with myosmine the increase of keto acid (104% of control) was even less but still significant whereas NNN- N-oxide and diol were significantly reduced to 72% and 79% of control, respectively. Our experiments with rats indicate significant mutual effects of some of the major tobacco alkaloids and most relevant TSNA. Further studies on the impact on smokers and the inhibitory effects of isothiocyanates are needed for a final risk assessment.

Incidences of Selected Lesions in Control Female Harlan Sprague–Dawley Rats from Two-Year Studies Performed by the National Toxicology Program

The NTP has a long history of using Fischer rats and has compiled a large database of incidences of lesions seen in control animals. Such a database is lacking for Harlan Sprague-Dawley (SD) rats. The intention of this paper is to report spontaneous lesions observed in female vehicle control Harlan SD rats, and to compare the incidence in 2 strains of rats (Fischer and Harlan SD) used in NTP studies. Female Harlan SD rats served as the test animals for a special series of 2-year studies. Male rats were not used in these studies. Complete histopathology was performed on all animals, and the pathology results underwent comprehensive NTP pathology peer review. The most commonly observed neoplasms in these female control Harlan SD rats were mammary gland fibroadenoma (71%), tumors of the pars distalis of the pituitary (41%) and thyroid gland C-cell tumors (30%). Female Fischer rats had incidences of 44% for mammary gland fibroadenomas, 34% for tumors of the pars distalis, and 16% for thyroid gland C-cell tumors. Fischer rats had a 15% incidence of clitoral gland tumors, while the Harlan SD rats had an incidence of < 1%. In contrast to Fischer F344 rats, the Harlan SD rats had a high incidence of squamous metaplasia of the uterus (44.2%). Squamous metaplasia is not a lesion commonly observed in NTP control Fischer rats. The Harlan SD rats had a very low incidence of mononuclear cell leukemia (0.5%), compared with an incidence of 24% in female Fischer rats.

Organ specificity of DNA adduct formation by tamoxifen and α-hydroxytamoxifen in the rat: implications for understanding the mechanism(s) of tamoxifen carcinogenicity and for human risk assessment

Tamoxifen is an anti-oestrogen widely used in the adjuvant therapy of breast cancer and is also used as a prophylactic to prevent the disease in high-risk women. An increased risk of endometrial cancer has been observed in both settings. In rats, tamoxifen potently induces liver carcinomas and also induces uterine tumours when given neonatally. It forms DNA adducts in rat liver via the formation of alpha-hydroxytamoxifen, the ultimately reactive form being generated by sulfotransferase. In order to investigate the formation of tamoxifen-derived DNA adducts in other rat tissues, female Fischer F344 or Sprague-Dawley rats were treated with tamoxifen or alpha-hydroxytamoxifen by gavage or by intraperitoneal injection, daily for 1, 4 or 7 days, and DNA adducts were detected by (32)P-postlabelling analysis. Tamoxifen formed DNA adducts in the liver but not in other tissues (uterus, stomach, kidney, spleen and colon). alpha-Hydroxytamoxifen also formed adducts at high levels in liver, but with the exception of single animals (1/8) in which a low level of adducts was detected in the stomach in one case, and in the kidney in the other; it also did not give rise to adducts in other tissues. The results suggest that tamoxifen is a genotoxic carcinogen in rat liver, but a non-genotoxic carcinogen in rat uterus, making it, uniquely, a carcinogen with more than one mechanism of action. Mutagenicity experiments conducted in Salmonella typhimurium strains expressing bacterial or human N,O-acetyltransferase did not provide evidence that either alpha-hydroxytamoxifen or alpha-hydroxy-N-desmethyltamoxifen undergoes metabolic activation by acetylation. The confinement of ST2A2, the isozyme of hydroxysteroid sulfotransferase that can activate the compounds, mainly to rat liver is the possible reason for the formation of ducts in the liver but not in other organs of the rat.

F-DIO obesity-prone rat is insulin resistant before obesity onset

We previously created a novel F-DIO rat strain derived by crossing rats selectively bred for the diet-induced obesity (DIO) phenotype with obesity-resistant Fischer F344 rats. The offspring retained the DIO phenotype through 3 backcrosses with F344 rats but also had exaggerated insulin responses to oral glucose before they became obese on a 31% fat high-energy (HE) diet. Here, we demonstrate that chow-fed rats from the subsequent randomly bred progeny required 57% lower glucose infusions to maintain euglycemia during a hyperinsulinemic clamp in association with 45% less insulin-induced hepatic glucose output inhibition and 80% lower insulin-induced glucose uptake than F344 rats. The DIO phenotype and exaggerated insulin response to oral glucose in the nonobese, chow-fed state persisted in the F6 generation. Also, compared with F344 rats, chow-fed F-DIO rats had 68% higher arcuate nucleus proopiomelanocortin mRNA expression which, unlike the increase in F344 rats, was decreased by 26% on HE diet. Further, F-DIO lateral hypothalamic orexin expression was 18% lower than in F344 rats and was increased rather than decreased by HE diet intake. Finally, both maternal obesity and 30% caloric restriction during the third week of gestation produced F-DIO offspring which were heavier and had higher leptin and insulin levels than lean F-DIO dam offspring. Third-gestational week dexamethasone also produced offspring with higher leptin and insulin levels but with lower body weight. Thus F-DIO rats represent a novel and potentially useful model for the study of DIO, insulin resistance, and perinatal factors that influence the development and persistence of obesity.

Intramyocardial microdepot injection increases the efficacy of skeletal myoblast transplantation

Recent progress in the field of cellular cardiomyoplasty has opened new prospects for the treatment of ischemic heart disease and currently moves from bench to bedside. The aim of the present study was to develop a novel cell delivery technique, reducing target tissue damage and improving cell dispersion and engraftment. In 30 male Fischer F344 rats an infarction of the left ventricle was generated by ligation of the left anterior descendent artery. Seven days after infarction, either 15 microdepots of 10 microl myoblast cell suspension (microdepot group) or culture medium (control group) were injected into the infarcted region using an automatic pressure injection device, or three depots of 50 microl myoblast cell suspension (macrodepot group) were injected using the standard surgical technique. Echocardiography was performed in all rats before and 6 weeks after cell injection. In all groups the perioperative mortality was below 20%. Six weeks after cell transplantation, a significant improvement of ejection fraction was seen in both myoblast treated groups compared to controls (macrodepot, microdepot, control; 53.7+/-11.9, 70.7+/-2.0, 39.1+/-6.4; P=0.026, P<0.001). The microdepot group showed a more decent improvement than the macrodepot group (70.7+/-2.0 vs. 53.7+/-11.9, P=0.013). In both treated groups, grafted myoblasts differentiated into multinucleated myotubes within host myocardium, however, the engraftment pattern was different and angiogenesis was enhanced in the microdepot group. Intramyocardial multisite pressure injection allows the safe and reliable transplantation of several myoblast microdepots into an infarcted myocardium and improves the efficacy of myoblast transplantation compared to the standard technique.

Single and multiple congenic strains for hydrocephalus in the H-Tx rat

The H-Tx rat has fetal-onset hydrocephalus with a complex mode of inheritance. Previously, quantitative trait locus mapping using a backcross with Fischer F344 rats demonstrated genetic loci significantly linked to hydrocephalus on Chromosomes 10, 11, and 17. Hydrocephalus was preferentially associated with heterozygous alleles on Chrs 10 and 11 and with homozygous alleles on Chr 17. This study aimed to determine the phenotypic contribution of each locus by constructing single and multiple congenic strains. Single congenic rats were constructed using Fischer F344 as the recipient strain and a marker-assisted protocol. The homozygous strains were maintained for eight generations and the brains examined for dilated ventricles indicative for hydrocephalus. No congenic rats had severe (overt) hydrocephalus. A few pups and a significant number of adults had mild disease. The incidence was significantly higher in the C10 and C17 congenic strains than in the nonhydrocephalic F344 strain. Breeding to F344 to make F.H-Tx C10 or C11 rats heterozygous for the hydrocephalus locus failed to produce progeny with severe disease. Both bicongenic and tricongenic rats of different genotype combinations were constructed by crossing congenic rats. None had severe disease but the frequency of mild hydrocephalus in adults was similar to congenic rats and significantly higher than in the F344 strain. Rats with severe hydrocephalus were recovered in low numbers when single congenic or bicongenic rats were crossed with the parental H-Tx strain. It is concluded that the genetic and epigenetic factors contributing to severe hydrocephalus in the H-Tx strain are more complex than originally anticipated.

Expression of Stromal Cell-Derived Factor-1 and of Its Receptor CXCR4 in Liver Regeneration from Oval Cells in Rat

Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway.