F344 Rats Research Articles

The Effect of Gestational PFOS Exposure on Metabolism in the F344 Rat

Perfluorooctanesulfonate (or PFOS) is a type of PFOA known as (per-and poly-fluorinated alkylated substance) which is a manufactured compound chemical made of a strong carbon-fluorine and sulfonic acid. Due to its hydrophobic and lipophobic properties, industries use it in food wrappers, fire-fighting foam, sweat-proof clothing, and household cleaning products. PFOS exposure is an endocrine disruptor that may induce Metabolic Syndrome (MetS), an association of disorders that increases the risk of significant health issues like heart disease, diabetes, and stroke. Gestational and lactational exposure to Perfluorooctanesulfonate (PFOS) in F344/Stm rats was hypothesized to induce metabolic dysfunction in both female and male offspring. Pregnant F344 rats were given a control diet or diet containing 15mg/kg or 30mg/kg of PFOS; measures of body weight and body composition through nuclear magnetic resonance (NMR) were performed, and tissue was measured at the euthanasia of the dams. Both female and male offspring exposed during pregnancy and lactation were subject to the same protocol with tissue collection at three ages. On both low and high doses of PFOS, the pregnant females showed a decreased body weight gain compared to the control group; males and females also showed decreased body weight at three weeks of age when weaned from the exposed female. At 16 weeks of age, the same patterns were not revealed. The results from NMR suggested a 21% decrease in fat percentage and a 23% decrease in fat for the high exposure in the dams. The offspring at three weeks old showed a reduction in fat mass, fat mass %, and fat-free % in both high and low-dose groups compared to the control group; on the other hand, at 16 weeks old, this trend is not shown. The results suggest that PFOS had a short-term metabolic effect on the offspring at the younger; however, at the adult age, they seem to recover their initial phenotypes. For future studies, it suggested the focus on underlying mechanisms responsible for the rodent's recovery. ASPIRE Program (R25HL168782) Medical College of Wisconsin. 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.

P2X7 receptor knockout does not alter renal function or prevent angiotensin II-induced kidney injury in F344 rats

P2X7 receptors (P2X7) mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow (RBF) and glomerular filtration rate (GFR), suggesting that P2X7 receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. Here, we generated a novel P2X7 global knockout (KO) rat on the F344 background, hypothesising enhanced RBF and protection from chronic angiotensin II (ANG II)-induced injury. CRISPR/Cas9 technology introduced an early stop codon into exon 2 of P2rx7. In male and female KO and WT rats, renal P2X7 expression was measured by RTqPCR (n=4-5/group) and western blotting (n=3/group). Bone marrow-derived macrophages (BMDM) were used to assess P2X7 function by measuring interleukin (IL)-1β release (ELISA) after stimulation with lipopolysaccharide (LPS) (1μg/mL, 4h) and ATP (3mM, 1h) (n=5-6/group). In vivo kidney function was assessed in anesthetized rats (thiopental, 50mg/kg, i.p.) at baseline and following serial arterial ligation to induce acute pressure natriuresis (n=9-10/group). Arterial blood pressure (BP) was measured directly, and urine was collected via tube cystotomy. RBF was measured using a Transonic Doppler flow probe placed around the main renal artery, and cortical and medullary flux by laser Doppler needle probes. GFR was determined by FITC-inulin clearance. Urinary sodium was measured by sodium-selective electrode analysis. In a separate experiment, BP was recorded over five consecutive days in conscious male KO and WT rats via implanted radiotelemetry devices (n=7-8/group). Finally, male KO and WT rats (n=9-10/group) were infused with ANG II (250ng/kg/min, s.c.) for 6 weeks. At end, one renal artery was isolated and vascular reactivity to phenylephrine, acetylcholine and sodium nitroprusside determined by wire myography. Bladder urine was collected to measure albumin/creatinine ratio (n=4-7/group), and kidneys were harvested for histological analyses (n=6/group). Data were analysed with unpaired t-test (two groups compared) or two-way ANOVA (two variables), with p<0.05 statistically significant. P2rx7 mutation was confirmed by genome sequencing (Sanger). P2rx7 mRNA abundance was partially decreased in kidney from male (-54%) and female (-25%) KO vs WT. P2X7 protein was detected in kidneys from WT but not from KO rats. In BMDM from WT rats, LPS+ATP stimulation induced IL-1β release; this response was markedly suppressed in KO BMDM (males: 2577±140 vs 413±175pg/mL, p<0.0001; females: 1889±394 vs 265±121pg/mL, p<0.0001). In vivo BP, renal haemodynamics and tubular sodium handling were not different between genotypes for either sex (P>0.05). Radiotelemetry confirmed no difference in BP between KO and WT rats. Following chronic ANG II infusion, KO and WT rats had no renal artery dysfunction but showed similar increases in albuminuria (P=0.3587), renal perivascular fibrosis (Picrosirius Red, P=0.5393), cortical interstitium CD68-positive area (p=0.6546) and tubular casts (Periodic acid-Schiff, P=0.8803), compared with naïve controls. Contrary to our hypothesis, global genetic deletion of P2X7 did not affect renal hemodynamics and we found no significant role for P2X7 in the modulation of tubular sodium reabsorption. Our data do not support a major role of P2X7 in causing renal vascular and tubular injury. This work was supported by Diabetes UK grant 17/0005685, The British Heart Foundation(FS/15/60/31510; RE/18/5/34216), the Medical Research Council (MR/S01053X), and the University of Edinburgh College of Medicine & Veterinary Medicine PhD Studentship. 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.

Aging Reduces Cardiac Contractility by Altering Cardiac Regulatory and Contractile Proteins

Aging is associated with an increased incidence of cardiac dysfunction. We hypothesized that aging associated changes in cardiac regulatory and contractile proteins produce contractile dysfunction. We assessed regulatory and contractile protein expression as well as contractility in left ventricular cardiac muscle from Fischer F-344 rats at 6-, 18-, and 24-month of age. Age-related changes in the expression of β-MHC as well as the expression and phosphorylation of troponin I (TnI) and cardiac myosin binding C protein (MyBP-C) were assessed by Western blot. Aging was associated with an increase in the expression of β-MHC and the phosphorylation of both TnI at Ser23/24 and MyBP-C. Mechanical properties were assessed in Triton-X permeabilized anterior papillary muscle from the left ventricle from both 6- and 24-month old rats. In papillary muscle of 24-month old rats, there was a decrease in maximum Ca2+ activated force, Ca2+ sensitivity of force and the rate constant for force recovery after quick release (ktr). Additionally, cardiomyocytes were isolated from the left ventricle at 6-, 18-, and 24-month of age using a modified Langendorff system. Cardiomyocytes were then loaded with fura-2 AM to measure cytosolic calcium ([Ca2+]cyt) responses to electrical field stimulation (0.5 Hz). [Ca2+]cyt and contractile (sarcomere length shortening) responses to stimulation were simultaneously measured using an IonOptix system. In older cardiomyocytes, the amplitude of evoked [Ca2+]cyt responses was reduced while there was no difference in the kinetics of the [Ca2+]cyt transient. The extent of sarcomere length shortening as well as the rates of both shortening and relaxation were reduced. These results demonstrate that aging is associated with changes in the expression and phosphorylation of contractile and regulatory proteins, which produce changes in cardiac contractility. Supported by a Mayo CV Prospective Grant (FVB). 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.

Acute Peripheral Irisin Administration Increases Hippocampal Mass in Young, Healthy Rat Cohort

Introduction: Skeletal muscle-brain crosstalk is highlighted as an important contributor to the beneficial relationship between exercise and the brain. Irisin is a key crosstalk myokine due to the ability of irisin to cross the blood-brain barrier and mediate effects of exercise in the brain. Importantly, the influence of irisin in the brain is often demonstrated through chronic administration or supraphysiological dosing of irisin. The aim of the present study is to determine if an acute, non-supraphysiological dose of exogenous irisin is suffcient to induce previously established effects in the hippocampi of young, healthy rats. Methods: Twenty-four 2–3-month-old male and female F344 rats were divided into two groups of 12 (50% male/female) which either received an irisin (100 μg/kg) or vehicle intraperitoneal injection. Forty-eight hours following irisin injections, brains were sectioned and weighed. Levels of total and phosphorylated hippocampal cyclic-AMP Response Element Binding Protein (CREB), a downstream target of irisin signaling, were quantified with Jess Simple Western automated western blot analysis. Values are mean ± standard deviation. Results: Irisin administration resulted in greater hippocampi mass (relative to brain mass, %) compared to vehicle treated animals (3.65±0.24 vs 3.28±0.32%, p<0.01). Further, pCREB/CREB in the hippocampus of irisin animals (0.58±0.13) was 24.6% greater than vehicle (0.47±0.09, p<0.05). Irisin administration did not influence body mass or whole brain mass relative to body mass. Conclusions: The present study demonstrates that a non-supraphysiological dose of irisin is suffcient to increase hippocampal mass and CREB phosphorylation 48 hours post-injection. This work supports irisin as an important, physiologically relevant mediator of the beneficial relationship between exercise, skeletal muscle, and the brain. NIH P20GM113109. 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.

P2X7 receptor knockout does not alter renal function or prevent angiotensin II-induced kidney injury in F344 rats

P2X7 receptors mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow and filtration rate, suggesting that receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. We generated a P2X7 knockout rat on F344 background, hypothesising enhanced renal blood flow and protection from angiotensin-II-induced renal injury. CRISPR/Cas9 introduced an early stop codon into exon 2 of P2rx7, abolishing P2X7 protein in kidney and reducing P2rx7 mRNA abundance by ~ 60% in bone-marrow derived macrophages. The M1 polarisation response to lipopolysaccharide was unaffected but P2X7 receptor knockout suppressed ATP-induced IL-1β release. In male knockout rats, acetylcholine-induced dilation of the renal artery ex vivo was diminished but not the response to nitroprusside. Renal function in male and female knockout rats was not different from wild-type. Finally, in male rats infused with angiotensin-II for 6 weeks, P2X7 knockout did not reduce albuminuria, tubular injury, renal macrophage accrual, and renal perivascular fibrosis. Contrary to our hypothesis, global P2X7 knockout had no impact on in vivo renal hemodynamics. Our study does not indicate a major role for P2X7 receptor activation in renal vascular injury.

Proteins Involved in Focal Cell Adhesion and Podosome Formation Are Differentially Expressed during Colorectal Tumorigenesis in AOM-Treated Rats.

Colorectal tumorigenesis involves the development of aberrant crypt foci (ACF) or preneoplastic lesions, representing the earliest morphological lesion visible in colon cancer. The purpose of this study was to determine changes in protein expression in carcinogen-induced ACF as they mature and transform into adenomas. Protein expression profiles of azoxymethane (AOM)-induced F344 rat colon ACF and adenomas were compared at four time points, 4 (control), 8, 16, and 24 weeks post AOM administration (n = 9/group), with time points correlating with induction and transformation events. At each time point, micro-dissected ACF and/or adenoma tissues were analyzed across multiple quantitative two-dimensional (2D-DIGE) gels using a Cy-dye labeling technique and a pooled internal standard to quantify expression changes with statistical confidence. Western blot and subsequent network pathway mapping were used to confirm and elucidate differentially expressed (p ≤ 0.05) proteins, including changes in vinculin (Vcl; p = 0.007), scinderin (Scin; p = 0.02), and profilin (Pfn1; p = 0.01), By determining protein expression changes in ACF as they mature and transform into adenomas, a "baseline" of altered regulatory proteins associated with adenocarcinoma development in this model has been elucidated. These data will enable future studies aimed at biomarker identification and understanding the molecular biology of intestinal tumorigenesis and adenocarcinoma maturation under varying intestinal conditions.

Combination of Parenteral Amino Acid Infusion and Intermittent Loading Exercise Ameliorates Progression of Postoperative Sarcopenia in Rat Model.

Postoperative sarcopenia is associated with poor outcomes in hospitalized patients. However, few studies have focused on short-term postoperative sarcopenia. Furthermore, the influence of nutritional management using amino acids (AAs) comprising a peripheral parenteral nutrition (PPN) solution and its combination with exercise (Exc) is unclear. Hence, we established a postoperative sarcopenic rat model to evaluate the effects of parenteral AA infusion combined with Exc on skeletal muscles and investigate the underlying mechanisms involved in the amelioration of muscle atrophy. Male F344 rats underwent surgery followed by hindlimb suspension (HS) for 5 days. The rats were divided into AA (-), AA (+), AA (-)-Exc, and AA (+)-Exc groups. They were continuously administered a PPN solution with or without AA at 98 kcal/kg/day. The Exc groups were subjected to intermittent loading for 1 h per day. Postoperative sarcopenic rats exhibited decreased muscle strength and mass and an upregulated ubiquitin-proteasome system, autophagy-lysosome system, and fast-twitch fiber-related genes, especially in the AA (-) group. The AA (+)-Exc group exhibited attenuated decreased muscle strength, increased gastrocnemius mass, and a suppressed upregulation of muscle atrophy- and fast-twitch fiber-related genes. Therefore, parenteral AA infusion combined with Exc may be effective in preventing postoperative sarcopenia in hospitalized patients.

Sex-dependent responses to high concentration of binge ethanol in spleen of adolescent F344 rats.

We previously reported that binge ethanol induces atrophy of the spleen, a key immune organ, in adolescent male F344 rats. Because there are significant sex effects in immune function, we investigated whether binge ethanol exerts sex-dependent effects on the spleen, including producing splenic atrophy. We gave F344 rats ethanol (4.8 g/kg/day; 52% w/v; i.g.) on postnatal days [PND] 36 ~ 38 and sacrificed them on PND 39 for spleen collection. We performed immunophenotyping analysis of splenic cells and examined the expression of 158 genes related to alcohol metabolism, epigenetic modification, and immune regulation in the spleens of adolescent (PND 39) male and female rats. Following a 3-day ethanol exposure, a loss of body weight, and absolute and relative spleen weight, was seen only in male adolescent rats. Ethanol altered the relative proportions of lymphocyte subtypes in both sexes with different patterns. We also found that 3-day ethanol exposure induced sex-dependent gene expression changes in spleen. Among the 158 genes studied, the expression of only three genes was significantly increased in female rats. However, the expression of 30 genes was significantly increased/decreased in male rats. Female rats had greater expression of alcohol metabolizing enzyme genes in the spleen under physiological conditions and when stimulated by binge ethanol. The genes are involved in epigenetic modification were differentially expressed in a sex-dependent manner. We found that male adolescent rats were more sensitive to binge ethanol than female rats. Differential expression of the genes related to alcohol metabolism and epigenetic modification (of DNA methyltransferase and histone deacetylases) between the sexes could account for the observed sex-dependent responses to binge ethanol in adolescent rats.

Antagonizing Activin A/p15INK4b Signaling as Therapeutic Strategy for Liver Disease.

Activin A is involved in the pathogenesis of human liver diseases, but its therapeutic targeting is not fully explored. Here, we tested the effect of novel, highly specific small-molecule-based activin A antagonists (NUCC-474/555) in improving liver regeneration following partial hepatectomy and halting fibrosis progression in models of chronic liver diseases (CLDs). Cell toxicity of antagonists was determined in rat hepatocytes and Huh-7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Hepatocytes and hepatic stellate cells (HSCs) were treated with activin A and NUCC-555 and analyzed by reverse transcription-polymerase chain reaction and immunohistochemistry. Partial hepatectomized Fisher (F)344 rats were treated with NUCC-555, and bromodeoxyuridine (BrdU) incorporation was determined at 18/24/36/120/240 h. NUCC-555 was administered into thioacetamide- or carbon tetrachloride-treated F344 rats or C57BL/6 mice, and the fibrosis progression was studied. NUCC-474 showed higher cytotoxicity in cultured hepatic cells; therefore, NUCC-555 was used in subsequent studies. Activin A-stimulated overexpression of cell cycle-/senescence-related genes (e.g., p15INK4b, DEC1, Glb1) was near-completely reversed by NUCC-555 in hepatocytes. Activin A-mediated HSC activation was blocked by NUCC-555. In partial hepatectomized rats, antagonizing activin A signaling resulted in a 1.9-fold and 2.3-fold increase in BrdU+ cells at 18 and 24 h, respectively. Administration of NUCC-555 in rats and mice with progressing fibrosis significantly reduced collagen accumulation (7.9-fold), HSC activation indicated by reduced alpha smooth muscle actin+ and vimentin+ cells, and serum aminotransferase activity. Our studies demonstrate that activin A antagonist NUCC-555 promotes liver regeneration and halts fibrosis progression in CLD models, suggesting that blocking activin A signaling may represent a new approach to treating people with CLD.

Validation of putative biomarkers of furan exposure through quantitative analysis of furan metabolites in urine of F344 rats exposed to stable isotope labeled furan

Humans are chronically exposed to furan, a potent liver toxicant and carcinogen that occurs in a variety of heat-processed foods. Assessment of human exposure based on the furan content in foods is, however, subject to some uncertainty due to the high volatility of furan. Biomarker monitoring is thus considered an alternative or complementary approach to furan exposure assessment. Previous work suggested that urinary furan metabolites derived from the reaction of cis-2-butene-1,4-dial (BDA), the reactive intermediate of furan, with glutathione (GSH) or amino acids may serve as potential biomarkers of furan exposure. However, some metabolites were also reported to occur in urine of untreated animals, indicating either background contamination via animal feed or endogenous sources, which may limit their suitability as biomarkers of exposure. The overall aim of the present study was to accurately establish the correlation between external dose and concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed. To this end, the furan metabolites GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine), NAcLys-BDA (R-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid), NAcCys-BDA-NAcLys (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine) and NAcCys-BDA-NAcLys sulfoxide (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine sulfoxide) were simultaneously analyzed by stable isotope dilution ESI–LC–MS/MS as unlabeled and [13C4]-furan dependent metabolites following oral administration of a single oral dose of isotopically labelled [13C4]-furan (0.1, 1, 10, 100 and 1000 µg/kg bw) to male and female F344/DuCrl rats. Although a linear correlation between urinary excretion of [13C4]-furan-dependent metabolites was observed, analysis of unlabeled NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide revealed substantial, fairly constant urinary background levels throughout the course of the study. Analysis of furan in animal feed excluded feed as a source for these background levels. GSH-BDA was identified as the only furan metabolite without background occurrence, suggesting that it may present a specific biomarker to monitor external furan exposure. Studies in humans are now needed to establish if analysis of urinary GSH-BDA may provide reliable exposure estimates.

Network meta-analysis of the molecular mechanisms and signaling pathways underlying alcohol-induced thymic atrophy.

Thymic atrophy is characterized by loss of thymocytes, destruction of thymic architecture, and a subsequent decrease in naïve T cells with compromised immunity. Thymic atrophy occurs during aging. Environmental factors including alcohol misuse also induce thymic atrophy. Despite the link between alcohol misuse and thymic atrophy, the underlying mechanism is understudied. We aimed to identify molecules and signaling pathways that underly alcohol-induced thymic atrophy during aging. F344 rats were given 3-day binge-ethanol (4.8 g/kg/day; 52% w/v; i.g.) and the thymus was collected and weighed. Molecular mechanisms underlying ethanol-induced thymic atrophy were investigated by network meta-analysis using the QIAGEN Ingenuity Pathway Analysis (IPA). The molecules associated with ethanol were identified from the QIAGEN Knowledge Base (QKB) and those associated with thymic atrophy were identified from QKB and Mouse Genome Informatics (MGI). Aging-mediated Differential Expression Genes (DEGs) from mouse thymocytes were obtained from the Gene Expression Omnibus (GEO) database (GSE132136). The relationship between the molecules and associated signaling pathways were studied using IPA. Binge-ethanol decreased thymic weight in F344 rats. Our meta-analysis using IPA identified molecules commonly shared by ethanol and thymic atrophy through which simulation with ethanol increased thymic atrophy. We then obtained aging-mediated DEGs from the atrophied thymocytes. We found that ethanol contributed to thymic atrophy through modulation of the aging-mediated DEGs. Our network meta-analysis suggests that ethanol may augment thymic atrophy through increased expression of cytokines (e.g., IL-6, IL-17A and IL-33) along with their regulators (e.g., STAT1 and STAT3). Exposure to alcohol may augment thymic atrophy by altering the activity of key inflammatory mediators, such as STAT family members and inflammatory cytokines. These findings provide insights into the signaling pathways and upstream regulators that underly alcohol-induced thymic atrophyduring aging, suggesting that alcohol consumption could prepone thymic atrophy.

The Effects of Endoplasmic Reticulum Stress via Intratracheal Instillation of Water-Soluble Acrylic Acid Polymer on the Lungs of Rats.

Polyacrylic acid (PAA), an organic chemical, has been used as an intermediate in the manufacture of pharmaceuticals and cosmetics. It has been suggested recently that PAA has a high pulmonary inflammatory and fibrotic potential. Although endoplasmic reticulum stress is induced by various external and intracellular stimuli, there have been no reports examining the relationship between PAA-induced lung injury and endoplasmic reticulum stress. F344 rats were intratracheally instilled with dispersed PAA (molecular weight: 269,000) at low (0.5 mg/mL) and high (2.5 mg/mL) doses, and they were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months after exposure. PAA caused extensive inflammation and fibrotic changes in the lungs' histopathology over a month following instillation. Compared to the control group, the mRNA levels of endoplasmic reticulum stress markers Bip and Chop in BALF were significantly increased in the exposure group. In fluorescent immunostaining, both Bip and Chop exhibited co-localization with macrophages. Intratracheal instillation of PAA induced neutrophil inflammation and fibrosis in the rat lung, suggesting that PAA with molecular weight 269,000 may lead to pulmonary disorder. Furthermore, the presence of endoplasmic reticulum stress in macrophages was suggested to be involved in PAA-induced lung injury.

Abstract 7302: Effect of estrogen receptor modulator bazedoxifene on progression of carcinogen induced bladder cancer in female F344 rats

Abstract Bladder Cancer (BC) is the second most common genitourinary cancer with high recurrence and mortality rate due to metastatic muscle invasive BC (MIBC). An estimated 82,290 adults in the United States will be diagnosed with BC and one-fifth of the patients will die with this cancer in 2023. Sex disparity of BC is well known with 4 times higher risk in men, but women are more likely to die from it than men. Studies suggest that estrogen receptor (ER)-α and ER-β are over expressed in 18% and 63% of bladder tumors and associated with highly proliferative tumors and reduced overall survival. Hence, ER signaling is considered an important target for intercepting BC. Since the majority of BCs are non-invasive at diagnosis, developing agents that block BC progression may be beneficial for clinical translation. Bazedoxifene (BAZ), a clinically approved second generation SERM, is a co-repressor for both the ER-α and ER-β receptors, and it is a competitive inhibitor of 17-β-estradiol. In this study, BAZ was tested for toxicity and efficacy in an N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced BC rat model. Female F344 rats are administered carcinogen BBN (150mg x twice a week) for 8 weeks to induce BC. For toxicity evaluation, 5 doses of BAZ were fed (2.5 ppm to 12.5 ppm in diet) to BBN treated rats (n=6) for 10 weeks. After termination, serum, various organs, and bladder tissues were evaluated for toxicity biomarkers and histopathology. For efficacy study, Female F344 rats (8 weeks of age) were (n=24) administered BBN as stated above. A week later, rats received either control or BAZ (5 ppm and 10 ppm) containing diets for 33 weeks and terminated. Bladders and other major organs were analyzed grossly for size, weight, and visible abnormalities including tumors. Formalin fixed tumor sections were stained and histopathologically graded. Results showed that BBN-exposed rat bladders were significantly larger and heavier due to tumors (0.77±0.10g, Mean±SEM; p<0.0001) as compared to vehicle treated rats’ normal bladders (0.09±0.02g). Bladder tumor histopathology showed transitional cell carcinoma (TCC) and squamous cell carcinomas (SCC) with non-MIBC (NMIBC) and MIBC tumor grades in the vehicle control group. There was no significant difference in bladder weights between the 5 ppm and 10 ppm BAZ-treated vs. control group (p>0.05). However, histopathological evaluation showed ~20% (p>0.05) reduction in papilloma and NMIBC in the treated rats. Importantly, BAZ 5 ppm and 10 ppm treated rats had significantly less MIBC in a dose dependent manner (50%-70% less respectively; p<0.001) compared to control rats. In summary, our study demonstrates chemopreventive efficacy of bazedoxifene in interception of in BBN-induced rat bladder MIBC and warrants further investigation alone and in combination with complementary pathway targeting agents (Supported by NCI-PREVENT program 75N91019D00020-75N91020F00005). Citation Format: Venkateshwar Madka, Gopal Pathuri, Surya P. Singh, Srikanth Chiliveru, Anil Singh, Anh Bao, Nicole Stratton, Clinton J. Grubbs, Shizuko Sei, John Clifford, Brian Cholewa, Chinthalapally V. Rao. Effect of estrogen receptor modulator bazedoxifene on progression of carcinogen induced bladder cancer in female F344 rats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7302.

Abstract 2440: Cancer driver mutations as quantitative biomarkers of cancer risk interspecies analyses using CarcSeq

Abstract Unmet needs in cancer risk assessment include the ability to predict rodent life-time tumor responses from short-term exposures and a scientific basis for rodent to human extrapolation. As an approach to address these needs, the use of hotspot cancer driver mutations (CDMs) as biomarkers of cancer risk was integrated with an error-corrected, next generation sequencing (NGS) approach. We have developed and investigated the sensitivity of a high-throughput NGS method capable of detecting a panel of the most prevalent point mutations observed in human oncogenes and tumor suppressor genes. This method, CarcSeq, involves performing multiple, high-fidelity PCR reactions to amplify the most prevalent mutations reported to occur in human tumors, tagging the amplicons with 9 bp unique identifier sequences at each end, constructing libraries using Illumina ChIP Seq kits, sequencing the libraries on an Illumina NextSeq500 platform and generating single-strand consensus sequences (SSCS), bioinformatically, as a means of error-correction. We validated CarcSeq mutant fraction measurements through observations related to known cancer driver tissue-specificities and mutation spectra for normal human breast and lung, ductal carcinoma, and lung adenocarcinoma samples. Performance of a reconstruction experiment established CarcSeq has a sensitivity of 104. The approach has now been adapted to analyze analogous conserved codons in rat and mouse. For each of the three species, the developed panel includes 13-15 amplicons encompassing known cancer driver genes (e.g., KRAS, PIK3CA, and TP53) encompasses the equivalent of 30 known human hotspot codons and has a combined target sequence length of ~1 kb. Amplicons were generated from DNA isolated from 16-week-old, untreated rodent tissue samples with known differences in spontaneous tumor incidence: female Wistar, Sprague Dawley, and F344 rat mammary samples and male and female B6C3F1 and CD-1 mouse lung samples. The rodent panel was optimized to generate ~100,000 SSCS/amplicon. CDMs were observed at conserved codons in young, untreated rodents, and results are consistent with what was expected based on strain differences in spontaneous tumorigenesis. Citation Format: Kelly L. Harris, Binsheng Gong, Barbara Parsons, Jennifer Faske, Joshua Xu. Cancer driver mutations as quantitative biomarkers of cancer risk interspecies analyses using CarcSeq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2440.

Optimized rat models better mimic patients with irinotecan-induced severe diarrhea

Irinotecan-induced severe diarrhea (IISD) not only limits irinotecan’s application but also significantly affects patients’ quality of life. However, existing animal models often inadequately represent the dynamics of IISD development, progression, and resolution across multiple chemotherapy cycles, yielding non-reproducible and highly variable response with limited clinical translation. Our studies aim to establish a reproducible and validated IISD model that better mimics the pathophysiology progression observed in patients, enhancing translational potential. We investigated the impact of dosing regimens (including different dose, infusion time, and two cycles of irinotecan administration), sex, age, tumor-bearing conditions, and irinotecan formulation on the IISD incidence and severity in mice and rats. Lastly, we investigated above factors’ impact on pharmacokinetics of irinotecan, intestinal injury, and carboxylesterase activities. In summary, we successfully established a standard model establishment procedure for an optimized IISD model with highly reproducible severe diarrhea incidence rate (100%) and a low mortality rate (11%) in F344 rats. Additionally, the rats tolerated at least two cycles of irinotecan chemotherapy treatment. In contrast, the mouse model exhibited suboptimal IISD incidence rates (60%) and an extremely high mortality rate (100%). Notably, dosing regimen, age and tumor-bearing conditions of animals emerged as critical factors in IISD model establishment. In conclusion, our rat IISD model proves superior in mimicking pathophysiology progression and characteristics of IISD in patients, which stands as an effective tool for mechanism and efficacy studies in future chemotherapy-induced gut toxicity research.

Gene-dosage- and sex-dependent differences in the prodromal-Like phase of the F344tgHD rat model for Huntington disease.

In Huntington disease (HD) the prodromal phase has been increasingly investigated and is currently in focus for early interventional treatments. Also, the influence of sex on disease progression and severity in patients is under discussion, as a sex-specific impact has been reported in transgenic rodent models for HD. To this end, we have been studying these aspects in Sprague Dawley rats transgenic for HD. Here, we took up on the congenic F344tgHD rat model, expressing a fragmented Htt construct with 51 CAG repeats on an inbred F344 rat background and characterized potential sexual dimorphism and gene-dosage effects in rats during the pre-symptomatic phase (1-8 months of age). Our study comprises a longitudinal phenotyping of motor function, emotion and sensorimotor gating, as well as screening of metabolic parameters with classical and automated assays in combination with investigation of molecular HD hallmarks (striatal cell number and volume estimation, appearance of HTT aggregates). Differences between sexes became apparent during middle age, particularly in the motor and sensorimotor domains. Female individuals were generally more active, demonstrated different gait characteristics than males and less anxiolytic-like behavior. Alterations in both the time course and affected behavioral domains varied between male and female F344tgHD rats. First subtle behavioral anomalies were detected in transgenic F344tgHD rats prior to striatal MSN cell loss, revealing a prodromal-like phase in this model. Our findings demonstrate that the congenic F344tgHD rat model shows high face-validity, closely resembling the human disease's temporal progression, while having a relatively low number of CAG repeats, a slowly progressing pathology with a prodromal-like phase and a comparatively subtle phenotype. By differentiating the sexes regarding HD-related changes and characterizing the prodromal-like phase in this model, these findings provide a foundation for future treatment studies.

Quality not quantity: Deficient juvenile play experiences lead to altered medial prefrontal cortex neurons and sociocognitive skill deficits.

Reduced play experience over the juvenile period leads to adults with impoverished social skills and to anatomical and physiological aberrations of the neurons found in the medial prefrontal cortex (mPFC). Even rearing rats from high-playing strains with low-playing strains show these developmental consequences. In the present study, we evaluated whether low-playing rats benefit from being reared with higher playing peers. To test this, we reared male Fischer 344 rats (F344), typically thought to be a low-playing strain, with a Long-Evans (LE) peer, a relatively high-playing strain. As juveniles, F344 rats reared with LE rats experienced less play and lower quality play compared to those reared with another F344. As adults, the F344 rats reared with LE partners exhibited poorer social skills and the pyramidal neurons of their mPFC had larger dendritic arbors than F344 rats reared with same-strain peers. These findings show that being reared with a more playful partner does not improve developmental outcomes of F344 rats, rather the discordance in the play styles of F344 and LE rats leads to poorer outcomes.

Formation of hepatocyte cytoplasmic inclusions and their contribution to methylcarbamate-induced hepatocarcinogenesis in F344 rats.

Methylcarbamate (MC), a reaction product between dimethyl dicarbonate and ammonia or ammonium ion, is a potent hepatocarcinogen in F344 rats. Various genotoxicity tests have shown negative results for MC. Although previous studies have described the effects of MC on the liver, including the formation of characteristic basophilic cytoplasmic inclusions (CIs) in hepatocytes, the toxicological significance of CIs and their involvement in hepatocarcinogenesis remain unclear. In the current study, to elucidate the mechanisms of MC hepatocarcinogenesis, we examined hepatotoxicity and genotoxicity after 4 weeks of administration of MC using gpt delta rats with an F344 genetic background as a reporter gene transgenic animal model. Histopathologically, single-cell necrosis, karyomegaly, and the formation of CIs positive for Feulgen staining were observed in hepatocytes at the carcinogenic dose, demonstrating the hepatotoxicity of MC. CIs were also detected as large micronuclei in liver micronucleus tests but not in the bone marrow, suggesting that MC could cause chromosomal instability specifically in the livers of rats. Reporter gene mutation assays demonstrated that MC did not induce mutagenicity even in the liver. Immunofluorescence analyses revealed that CIs exhibited loss of nuclear envelope integrity, increased heterochromatinization, and accumulation of DNA damage. An increase in liver STING protein levels suggested an effect on the cyclic GMP-AMP synthase/stimulator of interferon genes innate immune pathway. Overall, these data demonstrated the possible occurrence of chromothripsis-like chromosomal rearrangements via CIs. Thus, the formation of CIs could be a crucial event in the early stage of MC-induced hepatocarcinogenesis in F344 rats.

Aging or chronic stress impairs working memory and modulates GABA and glutamate gene expression in prelimbic cortex.

The glucocorticoid (GC) hypothesis posits that effects of stress and dysregulated hypothalamic-pituitary-adrenal axis activity accumulate over the lifespan and contribute to impairment of neural function and cognition in advanced aging. The validity of the GC hypothesis is bolstered by a wealth of studies that investigate aging of the hippocampus and decline of associated mnemonic functions. The prefrontal cortex (PFC) mediates working memory which also decreases with age. While the PFC is susceptible to stress and GCs, few studies have formally assessed the application of the GC hypothesis to PFC aging and working memory. Using parallel behavioral and molecular approaches, we compared the effects of normal aging versus chronic variable stress (CVS) on working memory and expression of genes that encode for effectors of glutamate and GABA signaling in male F344 rats. Using an operant delayed match-to-sample test of PFC-dependent working memory, we determined that normal aging and CVS each significantly impaired mnemonic accuracy and reduced the total number of completed trials. We then determined that normal aging increased expression of Slc6a11, which encodes for GAT-3 GABA transporter expressed by astrocytes, in the prelimbic (PrL) subregion of the PFC. CVS increased PrL expression of genes associated with glutamatergic synapses: Grin2b that encodes the GluN2B subunit of NMDA receptor, Grm4 that encodes for metabotropic glutamate receptor 4 (mGluR4), and Plcb1 that encodes for phospholipase C beta 1, an intracellular signaling enzyme that transduces signaling of Group I mGluRs. Beyond the identification of specific genes that were differentially expressed between the PrL in normal aging or CVS, examination of Log2 fold-changes for all expressed glutamate and GABA genes revealed a positive association between molecular phenotypes of aging and CVS in the PrL but no association in the infralimbic subregion. Consistent with predictions of the GC hypothesis, PFC-dependent working memory and PrL glutamate/GABA gene expression demonstrate comparable sensitivity to aging and chronic stress. However, changes in expression of specific genes affiliated with regulation of extracellular GABA in normal aging vs. genes encoding for effectors of glutamatergic signaling during CVS suggest the presence of unique manifestations of imbalanced inhibitory and excitatory signaling in the PFC.

Cherries with Different Geographical Origins Regulate Neuroprotection in a Photoperiod-Dependent Manner in F344 Rats.

The photoperiod is the main environmental cue that drives seasonal adaptive responses in reproduction, behavior, and metabolism in seasonal animals. Increasing evidence suggests that (poly)phenols contained in fruits can also modulate seasonal rhythms. (Poly)phenol-rich diets are associated with an improvement in cognitive function and neuroprotection due to their anti-inflammatory and antioxidative properties. However, it is unknown whether cherries affect neuroprotection in a photoperiod-dependent manner. To test this, F344 rats were exposed to L6 (6 h light/day), L12 (12 h light/day) and L18 (18 h light/day) photoperiods and fed a standard chow diet supplemented with either a control, lyophilized cherry 1 or cherry 2 with distinctive phenolic hallmarks. Physiological parameters (body weight, eating pattern index (EPI), testosterone, T4/T3) and hypothalamic key genes (Dio2, Dio3, Raldh1 and Ghrh) were strongly regulated by the photoperiod and/or fruit consumption. Importantly, we show for the first time that neurotrophs (Bdnf, Sod1 and Gpx1) in the hippocampus are also regulated by the photoperiod. Furthermore, the consumption of cherry 2, which was richer in total flavonols, but not cherry 1, which was richer in total anthocyanins and flavanols, enhanced neuroprotection in the hippocampus. Our results show that the seasonal consumption of cherry with a specific phenolic composition plays an important role in the hippocampal activation of neuroprotection in a photoperiod-dependent manner.