Female Brown Norway Rats Research Articles

The use of PD-1 functional knockout rats to study idiosyncratic adverse reactions to nevirapine.

Idiosyncratic drug reactions (IDRs) are associated with significant patient morbidity/mortality and lead to considerable drug candidate attrition in drug development. Their idiosyncratic nature makes the study of IDRs difficult. In particular, nevirapine is associated with a relatively high risk of serious skin rash and liver injury. We previously found that nevirapine causes a similar skin rash in female Brown Norway rats, but these animals do not develop significant liver injury. Programmed cell death protein-1 (PD-1) is an immune checkpoint involved in immune tolerance, and anti-PD-1 antibodies have been used to treat cancer. However, they increase the risk of liver injury caused by co-administered drugs. We found that PD-1-/- mice are more susceptible to drug-induced liver injury, but PD-1-/- mice are not a good model for all drugs. In particular, they do not develop a skin rash when treated with nevirapine, at least in part because they lack the sulfotransferase in their skin that forms the reactive metabolite responsible for the rash. Therefore, we developed a PD-1 mutant (PD-1m/m) rat, with an excision in the ligand-binding domain of PD-1, to test whether nevirapine would cause a more serious skin rash in these animals. The PD-1m/m rat was based on a Sprague Dawley background, which has a lower incidence of skin rash than Brown Norway rats. The treated PD-1m/m rats developed more severe liver injury than PD-1-/- mice, but in contrast to expectations, they did not develop a skin rash. Functional knockouts provide a unique tool to study the mechanisms of IDRs.

Inhibition of the cysteinyl leukotriene pathways increases survival of RGCs and reduces microglial activation in ocular hypertension

Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 μm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13–15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.

Development and Characterization of an Allergic Asthma Rat Model for Interventional Studies.

Allergic asthma is one of the most common chronic diseases of the airways, however it still remains underdiagnosed and hence undertreated. Therefore, an allergic asthma rat model would be useful to be applied in future therapeutic strategy studies. The aim of the present study was to develop an objective model of allergic asthma in atopic rats that allows the induction and quantification of anaphylactic shock with quantitative variables. Female Brown Norway rats were intraperitoneally sensitized with ovalbumin (OVA), alum and Bordetella pertussis toxin and boosted a week later with OVA in alum. At day 28, all rats received an intranasal challenge with OVA. Anaphylactic response was accurately assessed by changes in motor activity and body temperature. Leukotriene concentration was determined in the bronchoalveolar lavage fluid (BALF), and total and IgE anti-OVA antibodies were quantified in blood and BALF samples. The asthmatic animals’ motility and body temperature were reduced after the shock for at least 20 h. The asthmatic animals developed anti-OVA IgE antibodies both in BALF and in serum. These results show an effective and relatively rapid model of allergic asthma in female Brown Norway rats that allows the quantification of the anaphylactic response.

Menopause exacerbates visual dysfunction in experimental glaucoma

Glaucoma is the leading cause of irreversible blindness worldwide. Recently, estrogen deficiencies caused by early menopause, alterations in estrogen signaling via mutations in estrogen receptors, and polymorphisms along estrogen metabolic pathways have all been linked to an increased risk of developing glaucoma. Here, we examined how menopause and age impact visual function and retinal structure in an experimental model of glaucoma. Young (3–4 months) and aged (9–10 months) female Brown Norway rats were divided into pre- and post-menopausal cohorts by surgically inducing menopause via ovariectomy (OVX). After six weeks, ocular hypertension (OHT) was induced unilaterally for a period of eight weeks. Four cohorts were successfully followed to eight weeks: young sham (n = 8), young OVX (n = 9), aged sham (n = 10), and aged OVX (n = 11) animals. Intraocular pressure (IOP) was monitored weekly in all groups. Prior to inducing OHT (baseline) and at four and eight weeks after inducing OHT, we assessed visual acuity via the optomotor response (OMR) and retinal structure using optical coherence tomography (OCT). OHT decreased the OMR in all cohorts. We found that spatial frequency thresholds decreased by 54% in OVX animals after OHT compared to sham animals after OHT, regardless of age (p < 0.001). We also found thinning of the retinal nerve fiber layer (RNFL) and loss of total retinal thickness after induction of OHT. Aged animals had more thinning of the RNFL and loss of total retinal thickness compared to young animals (p < 0.001). Overall, OHT caused significant changes in visual function and retinal structure. Observing that OVX in young and aged animals further decreased spatial frequency thresholds after OHT suggests that an estrogen deficiency may intensify visual impairment after OHT.

Hippocampal-dependent memory retention is unaffected by a T21 light–dark cycle in female Fischer brown Norway rats

ABSTRACT The present study investigated circadian rhythms and hippocampal-dependent memory in female Fischer brown Norway rats. As in past reports with female Long Evans rats, we hypothesized that six days of exposure to an unentrainable T21 light–dark cycle that occurred during distributed Morris water task training would not impair the retention of the acquired memory. As expected, the rats did not have impaired retention of the place memory acquired in the distributed Morris water task. We next, quantified circadian entrainment two months after exposure to the T21 cycle. As expected, measures of circadian rhythms suggested that the rats exposed to the T21 had since entrained to a 12:12 light–dark cycle. To confirm that the T21 paradigm induces free-running circadian rhythms in female Fischer brown Norway rats, the control animals were subjected to the same T21 paradigm and circadian rhythms were continuously measured. Signs of free-running circadian rhythms were present during T21 exposure such as delayed phase of entrainment, elongated period, and decreased activity during the dark phase of the cycle. To our knowledge, this is one of the first characterization of activity rhythms in Fischer brown Norway rats.

Oxytocin improves probabilistic reversal learning but not effortful motivation in Brown Norway rats

Deficits in cognition and motivation are common and debilitating aspects of psychiatric disorders, yet still go largely untreated. The neuropeptide oxytocin (OT) is a potential novel therapeutic for deficits in social cognition and motivation in psychiatric patients. However, the effects of OT on clinically relevant domains of non-social cognition and motivation remain under studied. The present study investigated the effects of acute and chronic (21-day) administration of subcutaneous OT (0.04, 0.2, and 1 mg/kg) in cross-species translatable operant paradigms of reward learning and effortful motivation in male and female Brown Norway (BN) rats (n = 8–10/group). Reward learning was assessed using the probabilistic reversal learning task (PRLT) and effortful motivation was measured using the progressive ratio breakpoint task (PRBT). As predicted, BN rats exhibited baseline deficits in the detection of reversals of reward contingency in the PRLT relative to Long Evans (LE) rats. The two strains performed equally in the PRBT. Thirty minutes after a single OT injection (1 mg/kg), measures of both initial probabilistic learning (trials to first criterion) and subsequent reversal learning (contingency switches) were significantly improved to levels comparable with LE rats. The OT effect on switches persisted in male, but not female, BN rats 30 min, 24 h, and 6 days after long-term OT administration, suggesting the induction of neuroplastic changes. OT did not affect effortful motivation at any time-point. The beneficial effects of OT on reward learning in the absence of increased effortful motivation support the development of OT as a novel therapeutic to improve cognitive functioning.

Aging affects isoproterenol-induced water drinking, astrocyte density, and central neuronal activation in female Brown Norway rats.

Age-dependent impairments in the central control of compensatory responses to body fluid challenges have received scant experimental attention, especially in females. In the present study, we found that water drinking in response to β-adrenergic activation with isoproterenol (30 μg/kg, s.c.) was reduced by more than half in aged (25 mo) vs. young (5 mo) ovariectomized female Brown Norway rats. To determine whether this age-related decrease in water intake was accompanied by changes in central nervous system areas associated with fluid balance, we assessed astrocyte density and neuronal activation in the SFO, OVLT, SON, AP and NTS of these rats using immunohistochemical labeling for GFAP and c-fos, respectively. GFAP labeling intensity was increased in the SFO, AP, and NTS of aged females independent of treatment, and was increased in the OVLT of isoproterenol-treated rats independent of age. Fos immunolabeling in response to isoproterenol was reduced in both the SFO and the OVLT of aged females compared to young females, but was increased in the SON of female rats of both ages. Finally, fos labeling in the AP and caudal NTS of aged rats was elevated after vehicle control treatment and did not increase in response to isoproterenol as it did in young females. Thus, age-related declines in water drinking are accompanied by site-specific, age-related changes in astrocyte density and neuronal activation. We suggest that astrocyte density may alter the detection and/or processing of signals related to isoproterenol treatment, and thereby alter neuronal activation in areas associated with fluid balance.

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

ABSTRACTBody 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.

Effect of high-fructose and high-fat diets on pulmonary sensitivity, motor activity, and body composition of brown Norway rats exposed to ozone

Diet-induced obesity has been suggested to lead to increased susceptibility to air pollutants such as ozone (O3); however, there is little experimental evidence. Thirty day old male and female Brown Norway rats were fed a normal, high-fructose or high-fat diet for 12 weeks and then exposed to O3 (acute – air or 0.8 ppm O3 for 5 h, or subacute – air or 0.8 ppm O3 for 5 h/d 1 d/week for 4 weeks). Body composition was measured non-invasively using NMR. Ventilatory parameters and exploratory behavior were measured after the third week of subacute exposure. Bronchoalveolar lavage fluid (BALF) and blood chemistry data were collected 18 h after acute O3 and 18 h after the fourth week of subacute O3. The diets led to increased body fat in male but not female rats. O3-induced changes in ventilatory function were either unaffected or improved with the fructose and fat diets. O3-induced reduction in exploratory behavior was attenuated with fructose and fat diets in males and partially in females. O3 led to a significant decrease in body fat of males fed control diet but not the fructose or fat diet. O3 led to significant increases in BALF eosinophils, increase in albumin, and reductions in macrophages. Female rats appeared to be more affected than males to O3 regardless of diet. Overall, treatment with high-fructose and high-fat diets attenuated some O3 induced effects on pulmonary function, behavior, and metabolism. Exacerbation of toxicity was observed less frequently.

Drug-induced Liver Fibrosis: Testing Nevirapine in a Viral-like Liver Setting Using Histopathology, MALDI IMS, and Gene Expression.

Nevirapine (NVP) is associated with hepatotoxicity in 1-5% of patients. In rodent studies, NVP has been shown to cause hepatic enzyme induction, centrilobular hypertrophy, and skin rash in various rat strains but not liver toxicity. In an effort to understand whether NVP is metabolized differently in a transiently inflamed liver and whether a heightened immune response alters NVP-induced hepatic responses, female brown Norway rats were dosed with either vehicle or NVP alone (75 mg/kg/day for 15 days) or galactosamine alone (single intraperitoneal [ip] injection on day 7 to mimic viral hepatitis) or a combination of NVP (75/100/150 mg/kg/day for 15 days) and galactosamine (single 750 mg/kg ip on day 7). Livers were collected at necropsy for histopathology, matrix-assisted laser desorption/ionization imaging mass spectrometry and gene expression. Eight days after galactosamine, hepatic fibrosis was noted in rats dosed with the combination of NVP and galactosamine. No fibrosis occurred with NVP alone or galactosamine alone. Gene expression data suggested a viral-like response initiated by galactosamine via RNA sensors leading to apoptosis, toll-like receptor, and dendritic cell responses. These were exacerbated by NVP-induced growth factor, retinol, apoptosis, and periostin effects. This finding supports clinical reports warning against exacerbation of fibrosis by NVP in patients with hepatitis C.

Beneficial effect of chronic Staphylococcus aureus infection in a model of multiple sclerosis is mediated through the secretion of extracellular adherence protein

BackgroundBacterial infections have been assumed to worsen multiple sclerosis (MS) disease symptoms and to lead to increased neurodegeneration. However, the underlying biological mechanisms for these effects are complex and poorly understood. Here, we assessed the disease-modulating effects of chronic infection with Staphylococcus aureus, a common human pathogen, on the clinical course and the extent of neurodegeneration in experimental autoimmune encephalomyelitis (EAE), an animal model of MS.MethodsTo conduct this study, we established a persistent chronic infection in female brown Norway rats by inoculating Staphylococcus aureus (S. aureus) bacteria in a subcutaneously implanted tissue cages.ResultsIn this study, we observed that the introduction of a localized S. aureus infection during the subclinical phase of EAE induced a chronic systemic inflammatory response, consisting of increased T- and B-cell counts and systemic production of proinflammatory cytokines. Unexpectedly, the S. aureus infection completely prevented the development of clinical EAE, and markedly reduced inflammatory infiltration and demyelination of the optic nerve, while it increased the number of surviving retinal neurons. Using a S. aureus strain that lacked the extracellular adherence protein (Eap), we determined that the extracellular adherence protein is at least partially responsible for the inhibitory effect of S. aureus infection on autoimmune inflammation of the central nervous system.ConclusionsOur results demonstrate for the first time that chronic infection with S. aureus has a beneficial effect on EAE, indicating a dual role of infection in the pathogenesis of MS. We also showed that secretion of Eap by S. aureus plays a major role in preventing autoimmune inflammation of the CNS. Moreover, we identified Eap as a factor responsible for this protective effect.

Ketogenic Diet Attenuates NMDA-Induced Damage to Rat's Retinal Ganglion Cells in an Age-Dependent Manner

Objective: This study was conducted to investigate neuroprotective effects of a high fat/low carbohydrate and protein diet (ketogenic diet, KD) in a model of N-methyl <smlcap>D</smlcap>-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage in juvenile and young adult rats. Methods: Juvenile (30-35 days old) and young adult (56-70 days old) female Brown Norway rats were fed the KD for 21 days; rats exposed to a standard rodent diet (SRD) served as controls. The main constituents of the KD used in the present study were approximately 80% fats, 8% proteins, and less than 1% carbohydrates. On day 14 of exposure to the KD (or the SRD in the control group), each rat received a single intravitreal injection of NMDA; RGCs were then retrogradely labelled by hydroxystilbamidine on day 19 and collected on day 21 to assess the degree of damage induced by NMDA. Blood biomarkers to confirm the expected metabolic response to the KD (i.e. ketosis and hypoglycaemia) were also assessed. Results: Although both the juvenile and young adult rats developed comparable ketosis and hypoglycaemia when fed the KD, NMDA-induced loss in RGCs was significantly attenuated only in juvenile rats exposed to the KD in comparison with those fed the SRD; exposure to the KD had no protective effect in young adult rats. In summary, exposure to the KD had a neuroprotective effect in NMDA-induced RGC damage in juvenile rats, but not in young adult rats. Conclusion: These results support further exploration of metabolic interventions to treat optic neuropathies associated with neurodegeneration.

Strain differences in pH-sensitive K+ channel-expressing cells in chemosensory and nonchemosensory brain stem nuclei.

The ventilatory CO2 chemoreflex is inherently low in inbred Brown Norway (BN) rats compared with other strains, including inbred Dahl salt-sensitive (SS) rats. Since the brain stem expression of various pH-sensitive ion channels may be determinants of the CO2 chemoreflex, we tested the hypothesis that there would be fewer pH-sensitive K(+) channel-expressing cells in BN relative to SS rats within brain stem sites associated with respiratory chemoreception, such as the nucleus tractus solitarius (NTS), but not within the pre-Bötzinger complex region, nucleus ambiguus or the hypoglossal motor nucleus. Medullary sections (25 μm) from adult male and female BN and SS rats were stained with primary antibodies targeting TASK-1, Kv1.4, or Kir2.3 K(+) channels, and the total (Nissl-stained) and K(+) channel immunoreactive (-ir) cells counted. For both male and female rats, the numbers of K(+) channel-ir cells within the NTS were reduced in the BN compared with SS rats (P < 0.05), despite equal numbers of total NTS cells. In contrast, we found few differences in the numbers of K(+) channel-ir cells among the strains within the nucleus ambiguus, hypoglossal motor nucleus, or pre-Bötzinger complex regions in both male and female rats. However, there were no predicted functional mutations in each of the K(+) channels studied comparing genomic sequences among these strains. Thus we conclude that the relatively selective reductions in pH-sensitive K(+) channel-expressing cells in the NTS of male and female BN rats may contribute to their severely blunted ventilatory CO2 chemoreflex.

Intergenerational and parent of origin effects of maternal calorie restriction on Igf2 expression in the adult rat hippocampus

Insulin-like growth factor 2 (Igf2) regulates development, memory and adult neurogenesis in the hippocampus. Calorie restriction (CR) is known to modulate non-neuronal Igf2 expression intergenerationally, but its effect has not been evaluated on brain Igf2. Here, Sprague-Dawley (S) dams underwent moderate CR between gestational days 8-21. To identify parent of origin expression pattern of the imprinted Igf2 gene, their offspring (SS F1) were mated with naïve male or female Brown Norway (B) rats to obtain the second generation (BS and SB F2) progeny. CR did not affect adult hippocampal Igf2 transcript levels in SS F1 males or their BS F2 progeny, but increased it in SS F1 females and their SB F2 offspring. The preferentially maternal Igf2 expression in the SB F2 control male hippocampus relaxed to biallelic with CR, with no effect of grandmaternal diet in any other groups. Thus, allele-specific and total expression of hippocampal Igf2 is affected by maternal, grandmaternal CR in a strain and sex-specific manner.

Attenuation of the hypercapnic ventilatory response in the Brown Norway (BN) rat occurs prior to postnatal (P) day 26 and does not appear to be influenced by gender

Adult (&lt;49 days of age) inbred Brown Norway (BN) rats exhibit a blunted hypercapnic ventilatory response compared to inbred Dahl salt sensitive (SS) and outbred Sprague Dawley (SD) strains (Hodges et al., 2002), despite developing high CO2 sensitivity from post‐natal (P) day P12–14 until at least P20–21 (Davis et al., 2006). Thus, we hypothesized that CO2 sensitivity is attenuated between P30–P49 in the BN, but not the SS rat strain, and that the identification of a time window and/or potential gender difference may provide insights into the cause. The hypercapnic ventilatory response (FICO2=0.07) of male (n=6) and female (n=6) BN, and male SS (n=6) rats was measured with flow‐through plethysmography from P26–47, and expressed as a percentage of room air breathing (VE; % control). VE increased during hypercapnia in both male and female BN rats to (151%) at the earliest age studied (P26–27), and was similar throughout the remainder of the ages studied with one exception. At P30–31 there was a modest increase in the hypercapnic ventilatory response in male BN rats, but it was also highly variable. Additional comparisons to the SS strain await further analysis. We conclude from these data that the apparent attenuation of CO2 sensitivity in BN rats occurs prior to P26, and does not appear to be dependent upon gender. Supported by NIH HL097033.

Polycystic kidney disease gene in the Lewis polycystic kidney rat is mapped to chromosome 10q21&amp;ndash;q26

Correspondence: Jada Pasquale Yengkopiong Dr John Garang Memorial University of Science and Technology, Faculty of Science and Technology, Bor, Jonglei State, Republic of South Sudan Tel +211 955 621 320 Email jadalojuka@yahoo.com.au Background: Polycystic kidney disease (PKD) is a life-threatening disorder that affects the kidneys of millions of people across the world. The disease is normally inherited, but it can also be acquired, and leads to development of many cysts in the renal nephrons. In this study, the aim was to characterize PKD in the Lewis polycystic kidney (LPK) rat, the newest model for human PKD. Methods: Mating experiments were performed between male LPK rats with PKD and female Brown Norway and Wistar Kyoto rats without PKD to raise second filial (F2) and backcross 1 (BC1) progeny, respectively. Rats that developed PKD were identified. Histological examination of the kidneys and liver was performed. Liver tissue samples were collected from each rat and used to extract DNA. The extracted DNA was amplified, and mapping and linkage analyses were performed to identify the quantitative trait locus that controlled the disease phenotypes. Results: It was established that the disease was controlled by a recessive mutation in a single gene (F2: PKD = 42, non-PKD = 110, χ = 0.53; BC1: PKD = 67, non-PKD = 72, χ = 0.18, P . 0.05) and that the disease was inherited as autosomal recessive polycystic kidney disease (ARPKD). The rats with PKD developed larger fluid-filled cystic kidneys, higher systolic blood pressure, and anemia. However, there were no extrarenal cysts and no pup deaths. Mapping studies and linkage analyses associated the disease phenotypes in both the F2 and BC1 rats to chromosome 10q21–q26, giving a maximum LOD score of 7.9 (P = 0.00001) between peak markers D10Rat180 and D10Rat26. Conclusion: The quantitative trait locus on chromosome 10q21–q26 does not contain the Pkhd-1 gene, and it is different from quantitative trait loci that control ARPKD in other murine models. The candidate genes located in the quantitative trait locus are important in signal transduction, cell growth, cell proliferation, and cell differentiation. Although expression of PKD in the LPK rat shares common phenotypic traits with ARPKD caused by mutation in Pkhd-1, the gene responsible for ARPKD in the LPK rat is different and unknown, or a mechanism yet to be identified is responsible for development of the disease.

Bioactivation of nevirapine to a reactive quinone methide: implications for liver injury.

Nevirapine (NVP) treatment is associated with a significant incidence of liver injury. We developed an anti-NVP antiserum to determine the presence and pattern of covalent binding of NVP to mouse, rat, and human hepatic tissues. Covalent binding to hepatic microsomes from male C57BL/6 mice and male Brown Norway rats was detected on Western blots; the major protein had a mass of ∼55 kDa. Incubation of NVP with rat CYP3A1 and 2C11 or human CYP3A4 also led to covalent binding. Treatment of female Brown Norway rats or C57BL/6 mice with NVP led to extensive covalent binding to a wide range of proteins. Co-treatment with 1-aminobenzotriazole dramatically changed the pattern of binding. The covalent binding of 12-hydroxy-NVP, the pathway that leads to a skin rash, was much less than that of NVP, both in vitro and in vivo. An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP. These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group. Attempts were made to develop an animal model of NVP-induced liver injury in mice. There was a small increase in ALT in some NVP-treated male C57BL/6 mice at 3 weeks that resolved despite continued treatment. Male Cbl-b–/– mice dosed with NVP had an increase in ALT of >200 U/L, which also resolved despite continued treatment. Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal. This is a different pattern from the histology of NVP-induced liver injury in humans. This is the first study to report hepatic covalent binding of NVP and also liver injury in mice. It is likely that the quinone methide metabolite is responsible for NVP-induced liver injury.

Potential allergenicity research of Cry1C protein from genetically modified rice

With the development of genetically modified crops, there has been a growing interest in available approaches to assess the potential allergenicity of novel gene products. We were not sure whether Cry1C could induce allergy. We examined the protein with three other proteins to determine the potential allergenicity of Cry1C protein from genetically modified rice. Female Brown Norway (BN) rats received 0.1mg peanut agglutinin (PNA), 1mg potato acid phosphatase (PAP), 1mg ovalbumin (OVA) or 5mg purified Cry1C protein dissolved in 1mL water by daily gavage for 42days to test potential allergenicity. Ten days after the last gavage, rats were orally challenged with antigens, and physiologic and immunologic responses were studied. In contrast to sensitization with PNA, PAP and OVA Cry1C protein did not induce antigen-specific IgG2a in BN rats. Cytokine expression, serum IgE and histamine levels and the number of eosinophils and mast cells in the blood of Cry1C group rats were comparable to the control group rats, which were treated with water alone. As Cry1C did not show any allergenicity, we make the following conclusion that the protein could be safety used in rice or other plants.

Oxazolone (OXA) is a respiratory allergen in Brown Norway rats

Oxazolone (OXA) is a potent contact allergen in man, and it is used as a model Th1-allergen to test (Q)SAR's and screening assays for allergenic potential of chemicals. However, it elevates serum IgE levels and Thelper2 cytokines at relatively low doses in test animals, suggesting that it has also respiratory allergenic potential. The lack of human data on respiratory allergenic potential of OXA may be due to lack of significant inhalation exposure. Here, female Brown Norway rats (BN) were sensitized by two or five dermal applications of OXA at the same total dose of 3.75 mg. Controls received vehicle. All animals were challenged by inhalation to 45 mg/m 3 OXA on day 21 and necropsy was performed on day 22. All sensitized animals had increased serum IgE. OXA challenge decreased breathing frequency, and induced apnoeic breathing in the sensitized animals – a hallmark of respiratory allergy in our model. An exudative, granulocytic inflammation was observed primarily in the larynx of the sensitized and challenged rats. Microarray analysis of lung tissue, sampled 24 h after challenge, revealed upregulation of several genes and activation of Gene Ontology (GO) pathways, which resembled more closely those found previously in lung tissue of rats sensitized and challenged by the respiratory allergen trimellitic anhydride than by the contact allergen dinitrochlorobenzene. The results indicate that the contact allergen OXA can also be a respiratory allergen, provided that it is inhaled. Its use as a model contact sensitizer must be reconsidered.

Abstract 4378: Defining the role of mammary stem cells and progenitor cells in the development of estrogen-induced mammary cancer in the rat

Abstract Substantial data implicate both estrogens and progestins in the etiology of breast cancer. However, the mechanisms through which these steroid hormones elicit their effects on breast cancer risk have not been elucidated. The ACI rat serves as a unique model for investigating the mechanisms through which estrogens and progestins contribute to breast cancer development and for defining the genetic bases of inherited risk of breast cancer. When treated with physiologic levels of 17β-estradiol (E2), female ACI rats develop mammary cancer at an incidence that approaches 100%. Interestingly, rapid development of mammary cancer in E2 treated ACI rats is dependent upon progesterone. The mammary cancers that develop in E2 treated ACI rats are estrogen dependent, frequently exhibit non-random patterns of genome instability, and are invasive. Because each of these phenotypes is also commonly exhibited by breast cancers in humans, the ACI rat appears to be highly relevant to the study of breast cancer etiology. Whereas female ACI rats are highly susceptible to E2-induced mammary cancer, female Brown Norway (BN) rats are resistant. Genetic studies performed by our laboratory localized a total of six quantitative trait loci (QTL), designated Emca3 through Emca9, that account for a large fraction of inherited susceptibility in F2 progeny generated in intercrosses between ACI and BN rats. The purpose of this study is to test the hypothesis that ACI and BN rats exhibit differences in the responsiveness of their mammary stem cell (MaSCs) and/or progenitor cell (MaPCs) populations to E2 and/or progesterone. By using a combination of markers CD31, CD45, CD24, CD29 and fluorescence-activated cell sorting, we have identified four distinct populations of mammary cells. To quantify the numbers of MaSC and MaPC in these populations, we are evaluating the ability of the basal (Lin-CD24+CD29high) and luminal (Lin-CD24+CD29low) cell populations to form colonies when cultured in vitro and to generate mamamry gland outgrowths when transplanted into the interscapular fat pad of recipient syngenaic or (ACIxBN)F1 rats. Any observed differeces in MaSC and/or MaPC number or function will be evaluated further using a set of congenic rat strains that harbor BN alleles across an individual Emca locus on the ACI genetic background. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2011-4378