BN Strain Research Articles

Quercetin Prevents Hypertension in Dahl Salt-sensitive Rats F ed a High-salt Diet Through Balancing Endothelial Nitric Oxide Synthase and Sirtuin 1.

A high-salt diet is a leading dietary risk factor for elevated blood pressure and cardiovascular disease. Quercetin reportedly exhibits cardioprotective and antihypertensive therapeutic effects. The objective of this study is to examine the effect of quercetin on high-salt dietinduced elevated blood pressure in Dahl salt-sensitive (SS) rats and determine the underlying molecular mechanism. Rats of the Dahl SS and control SS-13 BN strains were separated into five groups, SS-13 BN rats fed a low-salt diet (BL group), SS-13 BN rats fed a high-salt diet (BH group), Dahl SS rats fed a low-salt diet (SL group), Dahl SS rats fed a high-salt diet (SH group), and SH rats treated with quercetin (SHQ group). Blood pressure was checked three weeks into the course of treatment, and biochemical markers in the urine and serum were examined. Additionally, western blot was done to evaluate the sirtuin 1 (SIRT1) and endothelial nitric oxide synthase (eNOS) expression levels. Immunohistochemical analysis was performed to verify SIRT1 levels. We demonstrated that a high-salt diet elevated blood pressure in both SS-13 BN and Dahl SS rats, and quercetin supplementation alleviated the altered blood pressure. Compared with the SH group, quercetin significantly elevated the protein expression of SIRT1 and eNOS. Immunohistochemistry results further confirmed that quercetin could improve the protein expression of SIRT1. Quercetin reduced blood pressure by enhancing the expression of SIRT1 and eNOS in Dahl SS rats fed a high-salt diet.

The effect of mitochondrial genome heterogeneity on voluntary wheel running activity in aged rats

Introduction: Variation in the response to exercise training is a widely accepted concept. A large portion of exercise response variability is attributable to genetic heritability, potentially due to inherited maternal mitochondrial characteristics. Importantly, humans exhibit a heterogenous mitochondrial genome. Unfortunately, many preclinical models used to investigate molecular transducers of exercise training are inbred rodent models that lack mitochondrial diversity, which has poor translatability to humans. Recently, a rat model has been generated to reflect human mitochondrial genome heterogeneity. Hence, we propose using this rat model to investigate the impact of mitochondrial genome heterogeneity on voluntary wheel running (VWR) activity. Objective: To investigate the effect of mitochondrial genome heterogeneity on VWR activity in aged rats. Hypothesis: VWR activity will differ between two rat strains with divergent mitochondrial genomes. Methodology: Male and female OKC-HETB and OKC-HETW rats were obtained from the Oklahoma City VA Medical Center. Briefly, the OKC-HETB and OKC-HETW rat strains were generated by a four-way cross strategy using four inbred strains (Brown Norway [BN], Fischer 344, Lewis, and Wistar Kyto [WKY]). Subsequent reciprocal crosses between males and females from the two F1 hybrids were made to generate genetically heterogeneous rats with mitochondrial genomes from either the BN (OKC-HETB) or WKY (OKC-HETW) parental strains. As a result, OKC-HETB and OKC-HETW rats have equal numbers of alleles yet divergent mitochondrial genomes. Endurance capacity and neuromuscular function in 18-to 20-month-old rats were assessed by treadmill running and rotarod protocols before and after 8 weeks of VWR. After post-testing, animals are euthanized at 21-24 months old, and tissues are collected. The mitochondrial function of obtained tissues will be analyzed via high-resolution respirometry, and protein levels will be quantified using immunohistochemistry and Western blotting. Results: Current baseline data shows female rats weighed less than males, which was not affected by strain. Although no strain effect on treadmill performance was observed, females ran longer than males during the treadmill test. However, this sex effect was lost within strain comparisons. There was no strain effect on rotarod performance as latency to fall times were similar between strains. However, OKCHetW rats showed some improved latency to falls over time. Female OKC-HETB rats had a longer latency to falls at baseline than their male counterparts, which became similar between the sexes over time. Conversely, female OKC-HETW rats developed longer latency to falls compared to their male counterparts over time. Currently, 2 weeks of VWR activity was highly variable among rats in the W stain but similar among rats in the B strain. Conclusions: Our preliminary findings suggest that mitochondrial genome heterogeneity does not impact baseline physical function outcomes such as endurance capacity and neuromuscular function in exercise naïve 18-20 months old rats. However, biological sex appears to have a greater influence. Currently, strain and sex did not affect 2-weeks of VWR activity. However, this study is currently ongoing using an 8-week VWR intervention. Importantly, this study will evaluate the impact of mitochondrial genome heterogeneity on VWR adaptations in young and aged rats. Wake Forest University School of Medicine Start-up Funds to Ahn. R21 AG072137 to Richardson, Stout, and Austad. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Addition of Bacillus subtilis BN Strain Probiotic on Chicken Eggs Quality

The addition of probiotics is one effort to increase the chicken's appetite. There are many probiotics that can be mixed into feed to help improve the digestive and reproductive systems, including Bacillus subtilis, which has the function of increasing egg quality, including increasing shell thickness, increasing egg volume, increasing egg yolk and white index. This reseacrh aims to find out the effect of probiotic administration of Bacillus subtilis BN Strain on shell thickness, white index and chicken yolk index. This research used 38-week-old layer laying hens as many as 24 heads, randomized into four treatments with eight replays. Based on the analysis of statistical data, the Analysis Of Variance (ANOVA) test showed no noticeable difference (p>0.05) to the thickness of the shell, white index or egg yolk index. The results showed that the addition of probiotic Bacillus subtilis BN Strain as many as 0.005 grams/kg of feed, 0.01 grams/kg of feed and 0.02 kg/kg of feed had no noticeable effect on the increase in egg shell thickness, egg white index and egg yolk index.

The Power of the Heterogeneous Stock Rat Founder Strains in Modeling Metabolic Disease.

Metabolic diseases are a host of complex conditions, including obesity, diabetes mellitus, and metabolic syndrome. Endocrine control systems (eg, adrenals, thyroid, gonads) are causally linked to metabolic health outcomes. N/NIH Heterogeneous Stock (HS) rats are a genetically heterogeneous outbred population developed for genetic studies of complex traits. Genetic mapping studies in adult HS rats identified loci associated with cardiometabolic risks, such as glucose intolerance, insulin resistance, and increased body mass index. This study determined underappreciated metabolic health traits and the associated endocrine glands within available substrains of the HS rat founders. We hypothesize that the genetic diversity of the HS rat founder strains causes a range of endocrine health conditions contributing to the diversity of cardiometabolic disease risks. ACI/EurMcwi, BN/NHsdMcwi, BUF/MnaMcwi, F344/StmMcwi, M520/NRrrcMcwi, and WKY/NCrl rats of both sexes were studied from birth until 13 weeks of age. Birth weight was recorded, body weight was measured weekly, metabolic characteristics were assessed, and blood and tissues were collected. Our data show wide variation in endocrine traits and metabolic health states in ACI, BN, BUF, F344, M520, and WKY rat strains. This is the first report to compare birth weight, resting metabolic rate, endocrine gland weight, hypothalamic-pituitary-thyroid axis hormones, and brown adipose tissue weight in these rat strains. Importantly, this work unveils new potential for the HS rat population to model early life adversity and adrenal and thyroid pathophysiology. The HS population likely inherited risk alleles for these strain-specific traits, making the HS rat a powerful model to investigate interventions on endocrine and metabolic health.

EFFECT OF PROBIOTIC ADMINISTRATION Bacillus subtilis BN Strain ON QUALITY OF CHICKEN EGGS

This reseacrh aims to find out the effect of probiotic administration of Bacillus subtilis BN Strain on shell thickness, white index and chicken yolk index. This research used 38-week-old layer laying hens as many as 24 heads, randomized into four treatments with eight replays. Treatment consists of P0 without the addition of probiotics, P1 with the addition of Bacillus subtilis BN Strain as many as 0.005 grams / kg of feed, P2 with the addition of Bacillus subtilis BN Strain as many as 0.01 grams / kg of feed and P3 with the addition of Bacillus subtilis BN Strain as many as 0.02 grams / kg of feed. Based on the analysis of statistical data, the Analysis Of Variance (ANOVA) test showed no noticeable difference (p>0.05) to the thickness of the shell, white index or egg yolk index. The results showed that the addition of probiotic Bacillus subtilis BN Strain as many as 0.005 grams/kg of feed, 0.01 grams/kg of feed and 0.02 kg/kg of feed had no noticeable effect on the increase in egg shell thickness, egg white index and egg yolk index.

A Genetically Heterogeneous Rat Model with Divergent MitochondrialGenomes.

We generated a genetically heterogenous rat model by a 4-way cross strategy using 4 inbred strains (Brown Norway [BN], Fischer 344 [F344], Lewis [LEW], and Wistar Kyoto [KY]) to provide investigators with a highly genetically diverse rat model from commercially available inbred rats. We made reciprocal crosses between males and females from the 2 F1 hybrids to generate genetically heterogeneous rats with mitochondrial genomes from either the BN (OKC-HETB, a.k.a "B" genotype) or WKY (OKC-HETW a.k.a "W" genotype) parental strains. These two mitochondrial genomes differ at 94 nucleotides, more akin to human mitochondrial genome diversity than that available in classical laboratory mouse strains. Body weights of the B and W genotypes were similar. However, mitochondrial genotype antagonistically affected grip strength and treadmill endurance in females only. In addition, mitochondrial genotype significantly affected multiple responses to a high-fat diet (HFD) and treatment with 17α-estradiol. Contrary to findings in mice in which males only are affected by 17α-estradiol supplementation, female rats fed a HFD beneficially responded to 17α-estradiol treatment as evidenced by declines in body mass, adiposity, and liver mass. Male rats, by contrast, differed in a mitochondrial genotype-specific manner, with only B males responding to 17α-estradiol treatment. Mitochondrial genotype and sex differences were also observed in features of brain-specific antioxidant response to a HFD and 17α-estradiol as shown by hippocampal levels of Sod2 acetylation, JNK, and FoxO3a. These results emphasize the importance of mitochondrial genotype in assessing responses to putative interventions in aging processes.

Attenuated Strain of Mycobacterium tuberculosis BN: Characteristics.

We evaluated the vaccine properties of a novel attenuated strain of M. tuberculosis BN (Mtb BN) and its impact on the gut microbiota in inbred female mice in comparison with a virulent strain Mtb H37Rv and a vaccine strain BCG. The Mtb BN strain demonstrated the highest anti-tuberculosis vaccine effect in I/St mice highly susceptible to tuberculosis infection and the same effect as BCG in mice of the recombinant strain B6.I-100 and in β2 microglobulin gene knockout mice. No adverse effects of the new Mtb BN strain on the gut microbiota of BALB/c mice were revealed. The virulent strain Mtb H37Rv and the vaccine strain BCG decreased the main indicators of normocenosis (Bifidobacterium spp., Bifidobacterium animalis subsp. lactis, Akkermansia, and Erysipelotrichaceae) and led to disappearance of Clostridium perfingens, E. coli, Pseudomonas spp., which contributed to reduction of species diversity and the development of dysbiosis.

Abstract P1-07-10: Estrogen-stimulated DNA damage in mammary epithelium varies among strains of rodents and women

Abstract Background: Estrogens play an important role in breast development, but prolonged exposures and higher levels have been associated with increased risk of breast cancer. Recently, estrogens and environmental estrogenic chemicals have been shown to promote the formation of estrogen receptor-mediated co-transcriptional R-loops that lead to DNA double strand breaks in breast cancer cell lines. However, the DNA damage mediated by the estrogen receptors in the breast epithelium may vary among individuals. Experimental Design: Strains of mice and rats were used to investigate the genetic basis for variation in estrogen-induced DNA damage. The BALB/c and C57BL/6 strains of mice were selected as they differ in susceptibility to mammary tumors (Majhi et al., 2021). Similarly, the ACI and BN strains of rats differ in their susceptibility to mammary tumors induced by estrogen exposure. The mice and rats were treated with 17β-estradiol (E2) for up to 28 days, then tissues were analyzed for γH2AX foci to assess the presence of DNA double strand breaks. Normal breast tissues from average-risk and high-risk patients were maintained in culture (patient-derived explant cultures or PDEs) and exposed to either control media or supplemented with 10nM E2, then analyzed for γH2AX foci. Results: Proliferative responses were similar in both BALB/c and C57BL/6 mouse strains. However, DNA damage and R loops were significantly higher in the mammary epithelium of BALB/c mice whereas only background levels were detected in C57BL/6 mice. The highest level of γH2AX foci was found colocalized in nuclei of cells that were positive for estrogen receptor alpha (ERα+). The E2-stimulated γH2AX in BALB/c mice was not associated with differences in estrogen receptor (Esr1) level or transcriptional responses in target genes Pgr, Areg or expression of Apobec3. Similarly, DNA damage was increased significantly in the mammary epithelium of mammary tumor-susceptible ACI rats treated with E2, but not in the tumor-resistant BN strain. The effect of E2 was also examined in humans using breast explant cultures. Levels of E2-induced γH2AX were significantly higher in tissues from donors undergoing prophylactic mastectomy due to familial risk compared to tissues from average risk women undergoing reduction mammoplasty. Among average risk donors, significant increases in γH2AX by E2 treatment was observed in only 1 of five donors. Conclusions: These data demonstrate that E2-induced DNA damage varies among strains of rodents and women. The levels of E2-stimulated DNA damage can provide a valuable diagnostic tool to identify subsets of individuals for whom estrogen exposure may be pathogenic in breast epithelial cells due to impaired DNA double strand break repair due to inherited risk alleles or epigenetic alterations. Citation Format: Karen A. Dunphy, Aman Sharma, Prabin Majhi, Sallie S Schneider, Giovanna M Crisi, Grace Makari-Judson, D Joseph Jerry. Estrogen-stimulated DNA damage in mammary epithelium varies among strains of rodents and women [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-10.

Bacillus subtilis BN strain promotes Th1 response via Toll-like receptor 2 in polarized mouse M1 macrophage.

Bacillus subtilis BN strain (BN strain) was isolated from natto, a traditional Japanese fermented soybean food product. The present study investigated the Th1 responses of the BN strain on a mouse macrophage cell line, J774.1. In cell cultures, the BN strain (spore cell cultured in Schaeffer's sporulation media) significantly increased the production of interleukin (IL-)12 protein. The BN strain induced the mRNA expression of M1 polarization genes, such as inducible nitric oxide synthase and IL-12p40 mRNA, and suppressed the mRNA expression of intracellular marker genes of M2 polarization, such as arginase 1 mRNA. The BN strain downregulated the mRNA expression of Toll-like receptor 4 (TLR4), while it upregulated the mRNA expression of TLR2, MyD88, and nuclear factor kappa B (NF-κB). The production of IL-12 protein induced by the BN strain was decreased by inhibitors of MyD88, NF-κB, and IκB kinase. Moreover, the production of IL-12 was strongly suppressed by neutralizing antibody against TLR2. These results suggest that the BN strain promotes Th1 response via TLR2 signal in mouse M1 macrophage. PRACTICAL APPLICATIONS: Bacillus subtilis is known to have beneficial effects for the host. B. subtilis BN stain (BN strain) was isolated from natto, a traditional Japanese fermented soybean food product. The effects of the BN strain on the Th1 response in macrophage cell cultures were investigated in this work. We found that the spore cells of BN strain promoted the production of Th1-type cytokine, and induced macrophage M1 polarization via Toll-like receptor 2. This study can serve as a significant reference for the development of functional food and feed with immunostimulatory effects. Over time, new food and feed products containing the BN strain may emerge, such as Juice, powder, and tablet.

Effects of location and season on productivity and productive adaptability of two parent stock exotic layer chicken strains in a warm-wet environment

Tropical commercial poultry industry is anchored on exotic strains which suffer varying degrees of productivity and adaptability problems. Adaptability is the ability to produce better or demonstrate least variation in productivity under multiple environments. The study was undertaken to examine the productivity and adaptability of Bevan Nera (IB) and Is a Brown (IB) parent stock strains under two locations and seasons. Data on weekly feed intake (FI g), cock weight (Cockwt, g), hen weight (Henwt, g), hen house production (HHP, %) and egg weight (Ewt, g) were collected from a commercial parent stock breeding farm in Ibadan, Nigeria, on the two strains. Data were subjected to general linear model analysis (GLM) procedures using SAS (1999), while mean separation was by bonferoni t-test (P=0.05). The statistical model was randomized complete block design (RCBD) in factorial. Factors of importance were Genotype (S), Location (L) as Fixed, and Season (S) as random. The effects of location on seasonal productivity and season on locational productivity for strains were 7.38, 4.96; and 4.99, 8.11 (%) for BN, IB respectively. GxL and GxLxS interactions were significant (P<0.043 and <0.013) for adaptability of strains. Locational adaptability indices were 48.59 and 51.41 while Seasonal adaptability indices were 63.32 and 36.68 (%) respectively for BN and IB strains.

Enhanced Crop Productivity and Sustainability by Using Native Phosphate Solubilizing Rhizobacteria in the Agriculture of Arid Zones

The importance of phosphate solubilizing rhizobacteria (PSB) has been well-document as an option for enhancing sustainable agriculture. As a particular group of plant growth promoting rhizobacteria (PGPR), PSB play an important role in the soil phosphorus cycle, increasing the bioavailability for growth and plant development. This study analyses the plant growth promoting effects of 5 strains (BN0009, BN0013, BN0015, BN0024, and BN0035) out of 180 isolated from Jarava frigida (Phil.) F.Rojas (Poaceae), a native grass from the Andean Atacama desert from North of Chile. The five bacterial isolated (BN strains) were identified as non-pathogenic Erwinia sp. and show a high phosphate solubilization capacity for Ca(PO4) ranging from 608.9 to 781.4 mg/L. Strains IAA production varies between 23.5 and 35.9 mg/L, siderophores, phosphatase (alkaline and acid) production was also observed, but none of the five isolated presented antagonism against plant pathogens Botrytis sp. and Sclerotinia sp. All isolates enhanced seed germination in Lactuca sativa and Solanum lycopersicum (excepting BN009). Additionally, all strains stimulated the early root elongation and seedling development in lettuce and tomato. Pot experiments displayed that BN0015, BN0024, and BN0035 significantly promote plant growth regarding root and leaf area, root and leaf weight, as well as leaf number compared with non-treated plants. In a field experiment with lettuce and two fertilization treatments (50 and 100% of the recommended crop fertilization), BN0024 application improved crop productivity compared to respective control. P content in plants with bacterial inoculations increased significantly compared to control in either fertilization treatment, suggesting an improved nutrient uptake. Also, lettuce with 50% fertilization and inoculation with BN0024 equate productivity with the control 100% fertilization. Finally, we discuss these results in the context of applicability to enhance the agroecosystem productivity in arid and semiarid zones.

Abstract 4165: Novel genetic rat models to identify factors that modulate cardiac and tumor radiation sensitivity

Abstract Purpose/Objectives: Over 50% of breast cancer patients receive radiation therapy, but radiation doses can be limited by normal tissue toxicity. Radiation therapy can improve breast cancer-specific survival, but cardiac morbidity can be increased in patients with left-sided tumors. We used rat genetic models to identify targets to improve the therapeutic ratio of radiation. We assessed the radiation responsiveness of mammary tumors and the heart in genetically similar consomic rats conducive to genetic mapping. Materials/Methods: Female SS rats and SS.BN3 consomic rats, which are genetically identical to SS rats except that chromosome 3 is inherited from the BN strain, have previously been shown to exhibit different vascular dynamics and breast tumor growth. Human MDA-MD-231 cells or syngeneic mammary tumor cells developed from DMBA-induced mammary tumors were implanted orthotopically into immunodeficient or immunocompetent SS and SS.BN3 rats, respectively, and tumors were treated locally with mock or 5x4 Gy. To examine cardiac toxicity, adult female SS and SS.BN3 rats received image-guided localized whole-heart radiation to a dose of 24 Gy or 9 Gy x 5 (AP and 2 lateral fields, weighted 1:1:1). Echocardiograms with strain analysis were performed at baseline, 3 months and 5 months. The Student's t-test was used to compare values. Results: The BN strain-derived genetic variant(s) on rat chromosome 3 is important for tumor radiation sensitivity. Tumors in SS.BN3 rats were significantly more radiosensitive than tumors in the parental SS strain. A supra-additive effect was seen with both tumor cell lines, with recurrence-free survival of 30% vs. 67% at 137 days in SS vs SS.BN3 rats (p=0.02) in xenografts, and recurrence-free survival of 9% vs. 100% at 141 days in SS vs. SS.BN3 rats (p<0.0001) in syngeneic tumors. The SS female rats that received 24 Gy exhibited enhanced cardiac toxicity compared to SS.BN3 rats, with larger pericardial effusions in SS vs SS.BN3 rats (p<0.05), significantly elevated end diastolic volume (EDV) and end systolic volume (ESV) at 5 months (EDV: 0.62 vs. 0.49 mL, p<0.01; ESV: 0.12 vs. 0.03 mL, p<0.01), and increased cardiac mortality (5/11 SS vs. 0/7 SS.BN3 rats). Fractionated heart radiation yielded similar results. Taken together, the SS.BN3 tumors are more sensitive to radiation, while the hearts of SS.BN3 rats are protected against radiation toxicity, when compared to the SS strain. Conclusions: These results demonstrate that genetic variants on rat chromosome 3 alter the sensitivity to radiation therapy, enhancing tumor responses to radiation and protecting the heart, thus improving the therapeutic ratio. Gene expression analysis and genetic mapping will be performed to identify the causative target(s). This project has the potential to enhance the effectiveness and toxicity profile of radiation therapy in breast cancer. Citation Format: Rachel A. Schlaak, Anne Frei, Aronne M. Schottstaedt, Brian L. Fish, Leanne Harmann, Tracy Gasperetti, Michael J. Flister, Meetha Medhora, Jennifer L. Strande, Carmen R. Bergom. Novel genetic rat models to identify factors that modulate cardiac and tumor radiation sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4165.

Abstract P2-11-18: The use of consomic animal models to identify genetic factors that modulate radiation-induced cardiac toxicity

Abstract Purpose/Objectives: Radiation therapy is used by more than 50% of breast cancer patients, but radiation doses can be limited by normal tissue side effects. For example, breast cancer radiation therapy can improve breast cancer-specific survival, but increase cardiac deaths in those with left-sided cancers. Identifying genetic factors that can enhance tumor radiation sensitivity while decreasing normal tissue toxicities has the potential to improve the therapeutic ratio of radiation therapy – leading to more cures and less long-term toxicities. The use of animal models with differing genetic backgrounds to assess radiation toxicity, followed by genetic mapping of radiosensitivity phenotypes, has the potential to identify new targets that can predict cardiac toxicity from radiation therapy. This project examines how genetic host factors alter normal tissue toxicity risks from breast cancer radiation. Materials/Methods: Inbred female SS rats and SS.BN3 consomic rats, that are genetically identical to SS rats except that chromosome 3 is inherited from the BN strain, have previously been shown to exhibit different vascular dynamics and breast tumor growth. For this study, adult female SS and SS.BN3 rats received image-guided whole heart radiation to a dose of 21 Gy (3 fields, AP and 2 laterals). Cardiac troponin was serially measured at 2, 6, and 12 weeks, and echocardiograms with strain analysis were performed at baseline and 3 months. The Student's t-test was used to compare values. Results: The SS female rats exhibited enhanced cardiac toxicity compared to SS.BN3 rats, with cardiac troponin levels elevated at 12 weeks (0.32 ng/ml vs.0.08 ng/ml for SS vs. SS.BN3, p=0.01), and moderate to severe pericardial effusions seen in 6 of 9 SS rats vs. 2 of 7 SS.BN3 rats. At 3 months post-radiation, echocardiograms revealed increased left ventricular posterior wall thickness at end diastole (LVPWd) in SS vs. SS.BN3 rats (0.25 vs. 0.20 cm, p=0.002) and increased left ventricular mass (LVM) in SS vs. SS.BN3 rats (1.54 vs. 1.28 g, p<0.001). Taken together, the SS female rats are more sensitive to cardiac irradiation than SS.BN3. Conclusions: These results demonstrate that genetic variant on rat chromosome 3 alter the radiosensitivity to single fraction cardiac radiation therapy. Gene expression analysis and genetic mapping will be performed to identify the causative target(s). These models will also be expanded to test whether similar results are seen with fractionated cardiac radiation therapy. This project has the potential to enhance the effectiveness and toxicity profile of radiation therapy in breast cancer. Citation Format: Bergom C, Schlaak R, Frei A, Fish BL, Harmann L, Gasperetti T, Schottstaedt AM, Flister MJ, Medhora M, Strande JL. The use of consomic animal models to identify genetic factors that modulate radiation-induced cardiac toxicity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-11-18.

Abstract B07: Utilizing consomic xenograft models to identify genetic variants in the tumor microenvironment that determine breast cancer radiation responses

Abstract Progress in elucidating the molecular basis of breast cancer has allowed for treatment breakthroughs such as anti-estrogen and Her2-targeted therapy. It has also shaped the approaches to both surgical and systemic therapy. However, no similar use of molecular information has been utilized to better direct the use of radiation therapy. The development of predictive tools for the radiosensitivity of tumors could allow for personally tailored radiation doses, with treatment de-escalation for radiosensitive tumors, or dose escalation or the use of adjunct treatments in the case of radioresistant tumors. Communication between malignant tumor cells and the tumor microenvironment (TME) underlies most aspects of tumor biology, including chemotherapy and radiation resistance. We have developed a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel and without cell-sorting. Here we utilize these unique techniques to identify genetic variants in the TME that can affect radiation sensitivity. In CXM, human triple negative breast cancer MDA-MD-231 cells are orthotopically implanted into immunodeficient (IL2Rγ-/-) consomic rat strains, which are rat strains in which an entire chromosome is introgressed into the isogenic background of another inbred strain by selective breeding. Because the strain backgrounds are different but the tumor cells are not varied, the observed changes in tumor progression are due to genetic differences in the non-malignant TME. We hypothesized that the tumors in SS.BN3 rats (identical to SS rats but with BN strain chromosome 3) would be more sensitive to radiation due to increased tumor vascularity via CD31 staining, and increased tumor blood volume capacity, as measured by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Our studies demonstrate differential responses to radiation in the CXM model comparing parental SS (IL2Rγ) rats to SS.BN3 (IL2Rγ) rats treated with fractionated radiation therapy (4 Gray x 3), with altered tumor growth kinetics and tumor recurrence rates. A difference was seen in time to 5-fold increase in tumor growth, with 44 vs. >130 days for SS versus SS.BN3 rats (supra-additive, p<0.05). There was a recurrence-free survival of 30% vs. 67% at 130 days, with a median time to recurrence of 57 days vs. time not reached (>130 days) in the SS versus SS.BN3 rats (p=0.02). These results suggest that genetic determinants in the TME affect the radiation sensitivity of genetically identical tumor cells. Using SSRS, we identified a number of candidates on rat chromosome 3 that may potentially influence radiation sensitivity by altering the tumor vasculature. Future studies will further dissect the pathways responsible for the changes in radiation sensitivity. Determining TME factors that affect the radiation sensitivity of tumors has the potential to allow for more tailored and effective radiation treatments in breast cancer. Citation Format: Carmen Bergom, Michael Straza, Amy Rymaszewski, Anne Frei, Angela Lemke, Shirng-Wern Tsaih, Howard Jacob, Michael J. Flister. Utilizing consomic xenograft models to identify genetic variants in the tumor microenvironment that determine breast cancer radiation responses. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B07.

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.

Effects of mtDNA in SHR-mtF344 versus SHR conplastic strains on reduced OXPHOS enzyme levels, insulin resistance, cardiac hypertrophy, and systolic dysfunction.

Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtDNA) haplotype groups represented by the BN, F344, LEW, and SHR strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. F344 mtDNA by comparing the SHR vs. SHR-mt(F344) conplastic strains that are genetically identical except for their mitochondrial genomes. Altogether 13 amino acid substitutions in protein coding genes, seven single nucleotide polymorphisms in tRNA genes, and 12 single nucleotide changes in rRNA genes were detected in F344 mtDNA compared with SHR mtDNA. Analysis of oxidative phosphorylation system (OXPHOS) in heart left ventricles (LV), muscle, and liver revealed reduced activity and content of several respiratory chain complexes in SHR-mt(F344) conplastic rats compared with the SHR strain. Lower function of OXPHOS in LV of conplastic rats was associated with significantly increased relative ventricular mass and reduced fractional shortening that was independent of blood pressure. In addition, conplastic rats exhibited reduced sensitivity of skeletal muscles to insulin action and impaired glucose tolerance. These results provide evidence that inherited alterations in mitochondrial genome, in the absence of variation in the nuclear genome and other confounding factors, predispose to insulin resistance, cardiac hypertrophy and systolic dysfunction.

Chromosome substitution modulates resistance to ischemia reperfusion injury in Brown Norway rats

Brown Norway rats (BN, BN/NHsdMcwi) are profoundly resistant to developing acute kidney injury (AKI) following ischemia reperfusion. To help define the genetic basis for this resistance, we used consomic rats, in which individual chromosomes from BN rats were placed into the genetic background of Dahl SS rats (SS, SS/JrHsdMcwi) to determine which chromosomes contain alleles contributing to protection from AKI. The parental strains had dramatically different sensitivity to ischemia reperfusion with plasma creatinine levels following 45 minutes of ischemia and 24 hours reperfusion of 4.1 and 1.3 mg/dl in SS and in BN, respectively. No consomic strain showed protection similar to the parental BN strain. Nine consomic strains (SS-7BN, SS-XBN, SS-8BN, SS-4BN, SS-15BN, SS-3BN, SS-10BN, SS-6BN, and SS-5BN) showed partial protection (plasma creatinine about 2.5-3.0 mg/dl), suggesting that multiple alleles contribute to the severity of AKI. In silico analysis was performed using disease ontology database terms and renal function quantitative trait loci from the rat genome database on the BN chromosomes giving partial protection from AKI. This tactic identified at least 36 candidate genes, with several previously linked to the pathophysiology of AKI. Thus, natural variants of these alleles or yet to be identified alleles on these chromosomes provide protection against AKI. These alleles may be potential modulators of AKI in susceptible patient populations.

Maternal allergen exposure reprograms the developmental lung transcriptome in atopic and normoresponsive rat pups

The "fetal origins hypothesis" argued that physiological changes consequent to in utero exposures ultimately contribute to disease susceptibility in later life. The dramatic increase in asthma prevalence is attributed to early exposures acting on preexisting asthma-susceptible genotypes. We showed previously that distinct transcriptome signatures distinguish the developmental respiratory phenotype of atopic (Brown Norway, BN) and normoresponsive (Lewis) rats. We aimed to determine whether maternal allergen exposure would influence asthma pathogenesis by reprogramming primary patterns of developmental lung gene expression. Postnatal offspring of dams sensitized to ovalbumin before mating and challenged during pregnancy were assessed for lung function, inflammatory biomarkers, and respiratory gene expression. Although maternal ovalbumin exposure resulted in characteristic features of an allergic response (bronchoalveolar lavage neutrophils, IgE, methacholine-induced lung resistance) in offspring of both strains, substantial strain-specific differences were observed in respiratory gene expression. Of 799 probes representing the top 5% of transcriptomic variation, only 112 (14%) were affected in both strains. Strain-specific gene signatures also exhibited marked differences in enrichment for gene ontologies, with immune regulation and cell proliferation being prominent in the BN strain, cell cycle and microtubule assembly gene sets in the Lewis strain. Multiple ovalbumin-specific probes in both strains were also differentially expressed in lymphoblastoid cell lines from human asthmatic vs. nonasthmatic sibling pairs. Our data point to the existence of distinct, genetically programmed responses to maternal exposures in developing lung. These different response patterns, if recapitulated in human fetal development, can contribute to long-term pulmonary health including interindividual susceptibility to asthma.

Nonsynonymous variants in mt-Nd2, mt-Nd4, and mt-Nd5 are linked to effects on oxidative phosphorylation and insulin sensitivity in rat conplastic strains

Common inbred strains of the laboratory rat can be divided into four different mitochondrial DNA haplotype groups represented by the SHR, BN, LEW, and F344 strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. LEW mitochondrial genomes by comparing the SHR to a new SHR conplastic strain, SHR-mt(LEW); these strains are genetically identical except for their mitochondrial genomes. Complete mitochondrial DNA (mtDNA) sequence analysis comparing the SHR and LEW strains revealed gene variants encoding amino acid substitutions limited to a single mitochondrial enzyme complex, NADH dehydrogenase (complex I), affecting subunits 2, 4, and 5. Two of the variants in the mt-Nd4 subunit gene are located close to variants known to be associated with exercise intolerance and diabetes mellitus in humans. No variants were found in tRNA or rRNA genes. These variants in mt-Nd2, mt-Nd4, and mt-Nd5 in the SHR-mt(LEW) conplastic strain were linked to reductions in oxidative and nonoxidative glucose metabolism in skeletal muscle. In addition, SHR-mt(LEW) conplastic rats showed increased serum nonesterified fatty acid levels and resistance to insulin stimulated incorporation of glucose into adipose tissue lipids. These results provide evidence that inherited variation in mitochondrial genes encoding respiratory chain complex I subunits, in the absence of variation in the nuclear genome and other confounding factors, can influence glucose and lipid metabolism when expressed on the nuclear genetic background of the SHR strain.

Genes for Difference in Eosinophilic Phenotype between MES and BN.MES-Cybames Rats Are on Chromosomes 9, 5, and 1

The Matsumoto Eosinophilia Shinshu (MES) rat strain develops hereditary blood eosinophilia due to the mutant Cyba(mes) gene. In contrast, BN.MES-Cyba(mes) congenic rats, in which the mutant Cyba(mes) gene introduced into the background of the BN strain, have a normal blood eosinophil level despite showing robust proliferation of eosinophils in the bone marrow. However, the congenic rats manifest focal necrosis with eosinophilic infiltration in the liver, a phenotype rarely observed in the original MES rat strain. To elucidate the genetic basis for the strain differences, (MES × BN.MES-Cyba(mes))F(2) rats were bred, and genetic analyses of phenotypes for eosinophilia were performed. Blood and bone marrow eosinophil levels in the F(2) rats showed broad distributions, suggesting that the traits were under the influence of multiple genes. Genetic association studies revealed that BN-derived marker loci on chromosomes 9 and 5 were responsible for the increase in eosinophil level in the bone marrow, decrease in blood eosinophil level, and the induction of focal necrosis with eosinophilic infiltration in the liver. The BN-derived allele of the marker gene on chromosome 1 was responsible for the decrease of both bone marrow and blood eosinophil levels. These data suggest the existence of genes characterizing/distinguishing the eosinophilic phenotypes of MES and BN.MES-Cyba(mes) on these chromosomes, and form the basis for positional cloning studies of the genes. These studies will advance the understanding of the mechanisms involved in eosinophil mobilization from the bone marrow and recruitment to the organs.