Quantitative Analysis of UDP-Glucuronosyltransferase Ugt1a and Ugt2b mRNA Expression in the Rat Liver and Small Intestine: Sex and Strain Differences.

UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of numerous endogenous and exogenous compounds to facilitate their excretion from the body. Because rats are commonly used in nonclinical studies, information regarding UGT species differences between rats and humans would be helpful for understanding human pharmacokinetics. In this study, we determined the absolute mRNA expressions of Ugt isoforms in the liver and small intestine of male and female Sprague-Dawley, Fischer 344, and Wistar rats. The sum of the mRNA levels of Ugt isoforms expressed in the liver was significantly (P < 0.005) higher than that in the small intestine regardless of the strain and sex. Ugt2b mRNA levels represented approximately 80% of total Ugt mRNA levels in the liver, whereas Ugt1a mRNA levels accounted for almost 90% in the small intestine. Ugt2b2 mRNA was specifically expressed in Wistar rat liver, resulting in 2-fold higher expression of total hepatic Ugt mRNA in Wistar rats than that in the other strains. Wistar rats showed prominently higher Ugt2b3 and Ugt2b8 mRNA levels in the small intestine than the other strains. The difference between sexes was remarkable with regard to hepatic Ugt1a10 in any of the strains, although slight differences between sexes were also observed in multiple Ugt isoforms. Taken together, this study revealed sex and strain differences in mRNA levels of rat Ugts. The data shown here would be useful for the selection of rat strains in nonclinical studies.

Effect of genetic strain and gender on age-related changes in body composition of the laboratory rat.

Body fat serves as a storage compartment for lipophilic pollutants and affects the pharmacokinetics of many toxic chemicals. Understanding how body fat varies with gender, strain, and age may be essential for development of experimental models to study mechanisms of toxicity. Nuclear magnetic resonance (NMR)-based analysis serves as a noninvasive means of assessing proportions of fat, lean, and fluid in rodents over their lifetime. The aim of this study was to track changes in body composition of male and female Long-Evans (LE), Sprague-Dawley (SD), Fischer (F334), and Brown Norway (BN) rats from postweaning over a >2-yr period. Percent fat of preweaned LE and SD rats was markedly higher compared to the other strains. LE and SD strains displayed marked increases in body fat from weaning to 8 mo of age. Postweaned F344 male and females showed relatively low levels of percent fat; however, at 2 yr of age percent fat of females was equal to that of SD and LE in females. BN rats showed the highest levels of lean tissue and lowest levels of fat. Percent fat of the BN strain rose at the slowest rate as they aged. Percent fluid was consistently higher in males for all strains. Females tended to have higher percent fat than males in LE, SD, and F344 strains. Assessing changes in body fat as well as lean and fluid of various strains of male and female rats over their lifetime may prove useful in many research endeavors, including pharmacokinetics of lipophilic toxicants, mechanisms underlying obesity, and metabolic disorders.

Responding in a test of decision-making under risk is under moderate genetic control in the rat.

BACKGROUND: Risk-taking, measured with laboratory tasks such as the Balloon Analog Risk Task (BART), is associated with real-life manifestations of risky behaviors, which may be an important component of inherited liability to alcohol use disorders. To identify genomic factors that influence these traits, the current study (i) characterized performance of a rodent version of the BART in multiple inbred rat strains, (ii) tested the degree to which performance was under genetic control, (iii) explored sex differences in performance, and (iv) evaluated the risk-taking behavior of F1 progeny of high-risk- and low-risk-taking strains to examine modes of inheritance. METHODS: Male and female rats (N = 100) from 5 inbred strains (Wistar-Furth, Fischer-344, Lewis, Spontaneously Hypertensive, Brown Norway) and Wistar-Furth × Fischer-344 hybrids were tested in the rat-BART, as well as in tests of locomotor activity, sucrose preference, and general motivation. RESULTS: About 55% of the variance in risk-taking behavior was attributable to heritable factors. The Fischer-344 strain was the most risk-taking and the most variable in responding. The mating of low-risk-taking Wistar-Furth and Fischer-344 rats produced progeny that behaved most like the Fischer-344 strain. Consistent with prior research in this laboratory (Jentsch et al., 2010), all rats were sensitive to changes in both risk and reinforcement parameters in the rat-BART; rats decreased voluntary risk-taking in the face of increasing risk and increased lever pressing when reinforcement probabilities were reduced. CONCLUSIONS: Our results endorse a moderately heritable pattern of risk-taking behavior in rats. The behavior of the hybrid progeny suggests a polygenic model with most gene effects transmitted by mode of dominant inheritance. The identification of high-risk and low-risk strains allows for isolation of quantitative trait loci associated with task performance and for probing the relationships between risk-taking and dimensions of alcohol use disorders.

Comprehensive microRNA-seq transcriptomic profiling across 11 organs, 4 ages, and 2 sexes of Fischer 344 rats.

Rat is one of the most widely-used models in chemical safety evaluation and biomedical research. However, the knowledge about its microRNA (miRNA) expression patterns across multiple organs and various developmental stages is still limited. Here, we constructed a comprehensive rat miRNA expression BodyMap using a diverse collection of 320 RNA samples from 11 organs of both sexes of juvenile, adolescent, adult and aged Fischer 344 rats with four biological replicates per group. Following the Illumina TruSeq Small RNA protocol, an average of 5.1 million 50 bp single-end reads was generated per sample, yielding a total of 1.6 billion reads. The quality of the resulting miRNA-seq data was deemed to be high from raw sequences, mapped sequences, and biological reproducibility. Importantly, aliquots of the same RNA samples have previously been used to construct the mRNA BodyMap. The currently presented miRNA-seq dataset along with the existing mRNA-seq dataset from the same RNA samples provides a unique resource for studying the expression characteristics of existing and novel miRNAs, and for integrative analysis of miRNA-mRNA interactions, thereby facilitating better utilization of rats for biomarker discovery.

Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization.

Rats are effective model animals and have contributed to the development of human medicine and basic research. However, the application of reproductive engineering techniques to rats is not as advanced compared with mice, and genome editing in rats has not been achieved using embryos obtained by in vitro fertilization (IVF). In this study, we conducted superovulation, IVF, and knock out and knock in using IVF rat embryos. We found that superovulation effectively occurred in the synchronized oestrus cycle and with anti-inhibin antiserum treatment in immature rats, including the Brown Norway rat, which is a very difficult rat strain to superovulate. Next, we collected superovulated oocytes under anaesthesia, and offspring derived from IVF embryos were obtained from all of the rat strains that we examined. When the tyrosinase gene was targeted by electroporation in these embryos, both alleles were disrupted with 100% efficiency. Furthermore, we conducted long DNA fragment knock in using adeno-associated virus and found that the knock-in litter was obtained with high efficiency (33.3-47.4%). Thus, in this study, we developed methods to allow the simple and efficient production of model rats.

Introgression of F344 rat genomic DNA on BB rat chromosome 4 generates diabetes-resistant lymphopenic BB rats.

Failure to express the Gimap5 protein is associated with lymphopenia (lyp) and linked to spontaneous diabetes in the diabetes-prone BioBreeding (BBDP) rat. Gimap5 is a member of seven related genes located within 150 Kb on rat chromosome 4. Congenic DR.(lyp/lyp) rats, where BBDP lyp was introgressed onto the diabetes-resistant BBDR background (BBDR.BBDP.(lyp/lyp)), all develop diabetes between 46 and 81 days of age (mean +/- SE, 61 +/- 1), whereas DR.(lyp/+) and DR.(+/+) rats are nonlymphopenic and diabetes resistant. In an intercross between F1(BBDP x F344) rats, we identified a rat with a recombination event on chromosome 4, allowing us to fix 33 Mb of F344 between D4Rat253 and D4Rhw6 in the congenic DR.lyp rat line. Gimap1 and Gimap5 were the only members of the Gimap family remaining homozygous for the BBDP allele. Offspring homozygous for the F344 allele (f/f) between D4Rat253 and D4Rhw6 were lymphopenic (85 of 85, 100%) but did not develop diabetes (0 of 85). During rescue of the recombination, 102 of 163 (63%) rats heterozygous (b/f) for the recombination developed diabetes between 52 and 222 days of age (88 +/- 3). Our data demonstrate that introgression of a 33-Mb region of the F344 genome, proximal to the mutated Gimap5 gene, renders the rat diabetes resistant despite being lymphopenic. Spontaneous diabetes in the BB rat may therefore be controlled, in part, by a diabetogenic factor(s), perhaps unrelated to the Gimap5 mutation on rat chromosome 4.

Genetic strain differences in platelet aggregation and thrombus formation of laboratory rats.

Rats are employed to investigate the role of platelets in thrombus formation under flow conditions in vivo and to evaluate the pre-clinical potential of antiplatelet drugs. While Wistar and Sprague-Dawley (SD) strains are commonly used in thrombosis models, a number of rat strains have been established. Each strain possesses genetically unique characteristics such as hypertension, hyperglycemia or hyperlipidemia. The appropriate selection of a strain might have advantages for physiological and pharmacological studies. Comparative investigation of platelet aggregation among laboratory strains of rats is useful for the development of thrombosis models. In the present study, platelet aggregation response in eight laboratory rat strains, ACI, Brown Norway (BN), Donryu, Fischer 344 (F344), LEW, SD, Wistar and WKAH, were compared. Considerable strain differences were observed in ADP-, collagen- and TRAP-induced platelet aggregation. SD and BN are high-platelet-aggregation strains, while F344 and ACI are low-response strains. In the arteriovenous shunt thrombosis model, SD formed larger thrombi than F344 and Wistar rats. In the FeCl(3)-induced thrombosis model with the carotid artery, the time to occlusion of SD was significantly shorter than of F344 and ACI rats. F344 and ACI rats had significantly increased bleeding times compared with SD rat. The present study demonstrates that there are considerable strain differences in platelet aggregation among laboratory rats, which reflect thrombus formation.

Activated K-ras and N-ras oncogenes in primary renal mesenchymal tumors induced in F344 rats by methyl(methoxymethyl)nitrosamine.

Administration of methyl(methoxymethyl)nitrosamine to newborn Fischer 344 rats results in the preferential induction of renal tumors arising from the mesenchymal component of the kidney. DNA from a significant proportion of these tumors was capable of transforming NIH/3T3 cells. This report describes the renal tumor model, the detection of two different ras transforming genes in the kidney tumors (the N-ras oncogene in 1 and K-ras oncogene in 10 kidney tumors) and the characterization of DNA sequences specifying the transformed phenotype.

Comparison of Allogeneic and Syngeneic Rat Glioma Models by Using MRI and Histopathologic Evaluation.

Research in neurooncology traditionally requires appropriate in vivo animal models, on which therapeutic strategies are tested before human trials are designed and proceed. Several reproducible animal experimental models, in which human physiologic conditions can be mimicked, are available for studying glioblastoma multiforme. In an ideal rat model, the tumor is of glial origin, grows in predictable and reproducible patterns, closely resembles human gliomas histopathologically, and is weakly or nonimmunogenic. In the current study, we used MRI and histopathologic evaluation to compare the most widely used allogeneic rat glioma model, C6-Wistar, with the F98-Fischer syngeneic rat glioma model in terms of percentage tumor growth or regression and growth rate. In vivo MRI demonstrated considerable variation in tumor volume and frequency between the 2 rat models despite the same stereotactic implantation technique. Faster and more reproducible glioma growth occurred in the immunoresponsive environment of the F98-Fischer model, because the immune response is minimized toward syngeneic cells. The marked inability of the C6-Wistar allogeneic system to generate a reproducible model and the episodes of spontaneous tumor regression with this system may have been due to the increased humoral and cellular immune responses after tumor implantation.

Lewis and Fischer 344 rats as a model for genetic differences in spatial learning and memory: Cocaine effects.

Lewis (LEW) and Fischer 344 (F344) rats are considered a model of genetic vulnerability to drug addiction. We previously showed important differences in spatial learning and memory between them, but in contrast with previous experiments demonstrating cocaine-induced enhanced learning in Morris water maze (MWM) highly demanding tasks, the eight-arm radial maze (RAM) performance was not modified either in LEW or F344 rats after chronic cocaine treatment. In the present work, chronically cocaine-treated LEW and F344 adult rats have been evaluated in learning and memory performance using the Y-maze, two RAM protocols that differ in difficulty, and a reversal protocol that tests cognitive flexibility. After one of the RAM protocols, we quantified dendritic spine density in hippocampal CA1 neurons and compared it to animals treated with cocaine but not submitted to RAM. LEW cocaine treated rats showed a better performance in the Y maze than their saline counterparts, an effect that was not evident in the F344 strain. F344 rats significantly took more time to learn the RAM task and made a greater number of errors than LEW animals in both protocols tested, whereas cocaine treatment induced deleterious effects in learning and memory in the highly difficult protocol. Moreover, hippocampal spine density was cocaine-modulated in LEW animals whereas no effects were found in F344 rats. We propose that differences in addictive-like behavior between LEW and F344 rats could be related to differences in hippocampal learning and memory processes that could be on the basis of individual vulnerability to cocaine addiction.

Lewis and Fischer 344 strain differences in alpha2-adrenoceptors and tyrosine hydroxylase expression.

Lewis and Fischer 344 (F344) rats differ in their pharmacological responses to a variety of drugs such as opioids, which has been partially attributed to differences in the endogenous opioid tone. Since opioid and alpha2-adrenergic mechanisms closely interact in nociception and substance abuse, a comparative study of the endogenous alpha2-adrenergic system in both inbred strains is of interest. Alpha-2 adrenoceptor subtypes and tyrosine hydroxylase, the rate-limiting enzyme of the catecholamine biosynthesis, were studied by Taqman RT-PCR analysis of gene expression in four brain areas of F344 and Lewis rats: hypothalamus, hippocampus, striatum and cortex. No differences were found in the mRNA levels of alpha2A- and alpha2C-adrenoceptors in any of the areas examined, however F344 rats exhibited lower levels of alpha2B-adrenoceptor transcripts in the hippocampus and higher levels in the hypothalamus. Tyrosine hydroxylase gene expression was found to be higher in hippocampus and striatum of F344 rats compared to Lewis, and a consistent 2-fold increase of the protein levels was detected by Western blots only in the case of the hippocampus. These results together with previous studies strongly suggest that the hippocampal noradrenergic activity of Lewis and F344 rats could be involved in their different responses to pain, stress and drug addiction.

Changes in submaxillary gland gene expression in F344 rats by multiple dosing of theophylline.

Multiple exposure to theophylline, a phosphodiesterase (PDE) inhibitor, induces acinar hypertrophy in the salivary gland. This study examined the effect of theophylline on the gene expression of secretory proteins and phosphodiesterases in the submaxillary gland. Male F344 rats received saline or theophylline (50 mg/kg) intraperitoneally for 4 days. The gene expressions for the secretory protein, cystatin S (CysS), and PDE subfamilies 3A and 4D in the submaxillary gland were quantified using RT-PCR. Theophylline exposure resulted in a sustained increase in mRNA expression for CysS and PDE3A, but PDE4D gene expression was unchanged. Our results suggest that submaxillary hypertrophy is primarily caused by the enhanced transcription of CysS, and that the transcription of each PDE subfamily gene is regulated differently.

The complete mitochondrial genome sequence and mutations of the lung cancer model inbred rat strain (Muridae; Rattus).

We reported the complete mitochondrial genome sequencing of an important Lung cancer model inbred rat strain for the first time. The total length of the mitogenome was 16,312 bp. It harbored 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 non-coding control region. The mutation sites were analyzed by comparing with the reference BN strain.

Inheritance of adrenal phenylethanolamine N-methyltransferase activity in the rat.

Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that catalyzes the S-adenosyl-L-methionine-dependent methylation of (-)norepinephrine to (-)epinephrine in the adrenal medulla. Adrenal PNMT activity is markedly different in two highly inbred rat strains; enzyme activity in the F344 strain is more than fivefold greater than that in the Buf strain. Initial characterization of the enzyme in the two inbred strains reveals evidence for catalytic and structural differences, as reflected in dissimilar Km values for the cosubstrate (S-adenosyl-L-methionine) and prominent differences in thermal inactivation curves. To assess adrenal PNMT activity in an F344 X Buf pedigree, we employed a statistical procedure to test for one- and two-locus hypotheses in the presence of within-class correlations due to cage or litter effects. The PNMT data in the pedigree are best accounted for by segregation at a simple major locus superimposed upon a polygenic background; data obtained from the biochemical studies suggest that the major locus is a structural gene locus.

Suppression of genetic resistance to bone marrow grafts and natural killer activity by administration of fat emulsion.

Graft rejection is one of the major obstacles to successful bone marrow transplantation (BMT). If resistance to marrow grafting could be avoided, BMT could be used widely in treatment of hematological and immunological disorders. There has been evidence that natural killer (NK) cells play a major role in genetic resistance to BMT and that macrophages are also involved in genetic resistance. Agents toxic to macrophages such as silica and carrageenan have been found to have a suppressive effect on genetic resistance to BMT. Parenteral fat emulsions are known to accumulate in macrophages and to impair various functions of macrophages and those of the reticuloendothelial system. We show here that the administration of a fat emulsion, Intralipos 20%, to recipient mice can suppress genetic resistance to bone marrow grafts and NK cell activity probably through the impairment of the macrophage function. The administration of the fat emulsion might be a new tactic in conditioning protocols for human BMT in the future.

Slc:Wistar outbred rats show close genetic similarity with F344 inbred rats.

Although Slc:Wistar rats are used widely in biomedical research as outbred rats, close similarities in growth curves, survival rates, and immunological and biochemical phenotypes have been reported between Slc:Wistar and F344 inbred rats. We reported previously that nine genetic variations that were fixed in Slc:Wistar rats had identical genotypes in F344 rats. Here, we examined the genetic characteristics of Slc:Wistar rats using 27 simple-sequence length polymorphism (SSLP) markers and compared them with other Wistar stocks available in Japan and with some F344 strains. Among 27 SSLP loci, 23 (85%) were fixed in the Slc:Wistar rats, which was the highest among the other Wistar stocks. The 23 fixed loci shared identical genotypes with corresponding loci in F344 rats. Further, the predominant allele types in the unfixed loci had allele frequencies as high as 80%, and these alleles were identical in the F344 rats. When the nine genetic variations reported previously are added, a total of 32 (89%) out of the 36 loci examined were fixed and identical in the Slc:Wistar and F344 rat genomes. These findings indicate the low genetic variation in Slc:Wistar rats and the high genetic similarity between the Slc:Wistar and F344 inbred rats. This study demonstrates the importance of characterizing outbred rats and the need to pay ample attention to the genetic characteristics the Slc:Wistar rats for their proper use.

Genetic differences in estrogen-induced deoxyribonucleic acid synthesis in the rat pituitary: correlations with pituitary tumor susceptibility.

Anterior pituitary nuclei isolated from rats were used in an in vitro DNA synthesis assay. The rate of in vitro synthesis was increased by prior in vivo estrogen treatment in a time-dependent and dose-dependent manner, and the observed increase was similar in magnitude to that measured by [3H] thymidine incorporation in whole tissue. In vitro DNA synthesis was sensitive to N-ethylmaleimide but resistant to dideoxythymidine triphosphate. Rates of pituitary DNA synthesis were compared in the Fischer 344 (F344) rat, a strain highly susceptible to diethylstilbestrol-induced pituitary tumors, and in the Holtzman rat, a strain resistant to rapid tumor induction by diethylstilbestrol. After chronic treatment (8 weeks) with estrogen, elevated DNA synthesis (2- to 3-fold) was observed in F344 pituitaries but not in Holtzman pituitaries. After short term (2-4 days) treatment with estrogen, however, DNA synthesis increased 2-fold in both Holtzman and F344 pituitary nuclei. The elevated DNA synthesis declined in the Holtzman strain after 5 or 6 days of additional estrogen treatment, but elevated DNA synthesis persisted in the F344 strain. These results suggest that the genetic difference is due to the lack of a proliferation control mechanism in the F344 strain that functions in the Holtzman strain to shut off estrogen-stimulated cell proliferation.

The effects of aging on neuron number in the rat superior olivary complex.

The superior olivary complex (SOC) is an auditory center in the brainstem involved in the processing of binaural information. The ability to localize sounds is dependent on the efficient processing of binaural signals by the SOC and other auditory centers. It has been demonstrated that sound localization ability is impaired in aged rats, and morphological changes in the SOC may contribute to this deficit. In the present study, neuron counts were done on three key nuclei of the SOC: the medial nucleus of the trapezoid body (MNTB), the lateral superior olivary nucleus (LSO), and the medial superior olivary nucleus (MSO) in groups of Fischer 344 rats aged 3, 12, 24, and 30 months. Neuron number remains stable between 3 and 30 months of age in the LSO and MSO, however, in the MNTB, neuron number is significantly reduced at 24 and 30 months of age. Neuron loss in the MNTB of 24-month-old Fischer 344 rats is not as heavy as that reported earlier in 24-month-old Sprague-Dawley rats (8% loss versus 34% loss), indicating a strain difference with regard to aging in the SOC.

Normal gene expression in male F344 rat nasal transitional and respiratory epithelium.

The nasal epithelium is an important target site for chemically-induced toxicity and carcinogenicity in rodents. Gene expression profiles were determined in order to provide normal baseline data for nasal transitional/respiratory epithelium from healthy rats. Cells lining the rat nasal passages were collected and gene expression analysis was performed using Clontech cDNA Rat Atlas 1.2 arrays (1185 genes). The percentages of genes within specific average expression ranges were 4.2% at 45,000-1000, 14.8% at 1000-200, 25.0% at 200-68, and 56.0% below 68. Nine out of a subset of ten genes were confirmed for relative signal intensity using quantitative real-time RT-PCR. The most highly expressed genes included those involved in phase I (e.g. cytochrome P450s) and phase II (e.g. glutathione S-transferases) xenobiotic metabolism, bioenergetics (e.g. cytochrome oxidase), osmotic balance (e.g. Na(+)/K(+) ATPase) and epithelial ionic homeostasis (e.g. ion channels). Such baseline data will contribute to further understanding the normal physiology of these cells and facilitate the interpretation of responses by the nasal epithelial cells to xenobiotic treatment or disease.

Quantitation of age-related mitochondrial DNA deletions in rat tissues shows that their pattern of accumulation differs from that of humans.

Three age-related mtDNA deletions were identified, and the competitive polymerase chain reaction (PCR) was used to quantitate their levels in different Fisher 344 rat tissues. Deletions that removed 4834; 13273; or 13415nt of the mitochondrial genome were shown to be associated with 16 (mtDNA4834), nine (mtDNA13273), or five (mtDNA13415) nt direct repeats, respectively. The mtDNA4834 deletion was detected in an age-related manner in all tissues screened; the mtDNA13415 deletion was detected in old heart, and in both young and old brain; and the mtDNA13273 deletion was only detected in old brain tissues. The mtDNA4834 deletion was found to be at its highest level in the liver (1.88x10-2%), followed by the brain (0.22x10-2%) and kidney (0.40x10-2%) of old animals. Much lower levels were observed in old heart (0.07x10-2%) and lung (0. 04x10-2%). This distribution of mtDNA deletions in old rat tissues is in contrast to work done in humans where age-related deletions are present at the highest levels in post-mitotic tissues with much lower levels in more mitotic tissues. An inverse relationship was observed between the level of mtDNA deletions and the size of the deleted region, since the mtDNA13415 deletion was present at about a 100-fold lower level (0.53x10-5%) than the smaller mtDNA4834 deletion in old heart tissue.