Male F344 Rats Research Articles

Repeated Intravenous Administration of Human Neural Stem Cells Producing Choline Acetyltransferase Exerts Anti-Aging Effects in Male F344 Rats.

Major features of aging might be progressive decreases in cognitive function and physical activity, in addition to withered appearance. Previously, we reported that the intracerebroventricular injection of human neural stem cells (NSCs named F3) encoded the choline acetyltransferase gene (F3.ChAT). The cells secreted acetylcholine and growth factors (GFs) and neurotrophic factors (NFs), thereby improving learning and memory function as well as the physical activity of aged animals. In this study, F344 rats (10 months old) were intravenously transplanted with F3 or F3.ChAT NSCs (1 × 106 cells) once a month to the 21st month of age. Their physical activity and cognitive function were investigated, and brain acetylcholine (ACh) and cholinergic and dopaminergic system markers were analyzed. Neuroprotective and neuroregenerative activities of stem cells were also confirmed by analyzing oxidative damages, neuronal skeletal protein, angiogenesis, brain and muscle weights, and proliferating host stem cells. Stem cells markedly improved both cognitive and physical functions, in parallel with the elevation in ACh levels in cerebrospinal fluid and muscles, in which F3.ChAT cells were more effective than F3 parental cells. Stem cell transplantation downregulated CCL11 and recovered GFs and NFs in the brain, leading to restoration of microtubule-associated protein 2 as well as functional markers of cholinergic and dopaminergic systems, along with neovascularization. Stem cells also restored muscular GFs and NFs, resulting in increased angiogenesis and muscle mass. In addition, stem cells enhanced antioxidative capacity, attenuating oxidative damage to the brain and muscles. The results indicate that NSCs encoding ChAT improve cognitive function and physical activity of aging animals by protecting and recovering functions of multiple organs, including cholinergic and dopaminergic systems, as well as muscles from oxidative injuries through secretion of ACh and GFs/NFs, increased antioxidant elements, and enhanced blood flow.

Intratracheal administration of cross-linked water-soluble acrylic acid polymer is associated with inducible bronchi-related lymphoid tissue formation and allergic inflammation.

In a Japanese chemical factory, lung diseases such as pneumoconiosis have been reported among workers handling cross-linked water-soluble acrylic acid polymers (CWAAP). Our previous study reported that a single intratracheal administration of CWAAP induces acute inflammation and fibrosis. In this study, we investigated the effects of multiple intratracheal administrations of CWAAP on inflammatory responses and pulmonary fibrosis along with inducible bronchus-associated lymphoid tissues (iBALT) formation, which is involved in allergic inflammation. Male F344 rats (190-200 g) received single or multiple intratracheal administrations of phosphate-buffered saline (PBS) or CWAAP. To assess inflammatory responses and pulmonary fibrosis, immunohistochemical and histological staining was performed. CD68, CD163, CD169, TGF-β, and collagen I positive cells/areas in the lungs of the CWAAP-group rats were significantly increased than those in the PBS group. Furthermore, the number of iBALT structures, CD4 + T cells, along with CD19, PAX5, IL-4, GATA-3, T-bet, and IgE-positive cells in the terminal bronchioles and blood vessels of the lungs were significantly increased in the CWAAP group. Moreover, pulmonary fibrosis, iBALT formation, and levels of specific IgG were significantly increased in rats who received multiple intratracheal administrations of CWAAP compared to those with single intratracheal administration. Multiple intratracheal administrations of CWAAP potentiated the classical fibrotic pathway (M2 macrophage-TGF-β-collagen I) more potently than single intratracheal administration. Furthermore, it was possible that iBALT was formed around terminal bronchioles and blood vessels and the number of immune cells was increased, resulting in enhanced allergic inflammation and pulmonary fibrosis.

Sarcopenia of the longitudinal tongue muscles in rats

The tongue is a muscular hydrostat, with lingual movements occurring during breathing, chewing, swallowing, vocalization, vomiting, coughing and grooming/sexual activities. In the elderly, reduced lingual dysfunction and weakness contribute to increased risks of obstructive sleep apnea and aspiration pneumonia. In Fischer 344 (F344) rats, a validated model of aging, hypoglossal motor neuron death is apparent, although there is no information regarding tongue strength. The intrinsic tongue muscles, the superior and inferior longitudinal, transversalis and verticalis exist in an interdigitated state. Recently, we established a method to measure the specific force of individual intrinsic tongue muscle, accounting for the tissue bulk that is not in the direction of uniaxial force. In the longitudinal muscles of 6- (n = 10), 18- (n = 9) and 24-month-old (n = 12) female and male F344 rats, we assessed specific force, fatigability, fiber type dependent cross-sectional area (CSA) and overall CSA. Muscle force and fatigue was assessed ex vivo using platinum plate simulation electrodes. Tongue muscles were frozen in melting isopentane, and transverse sections cut at 10 µm. Muscle fiber type was classified based on immunoreactivity to myosin heavy chain (MyHC) isoform antibodies. In H&E stained muscle, CSA and uniaxial muscle contributions to total tongue bulk was assessed. We observed a robust ∼30% loss of longitudinal specific force, with reductions in overall longitudinal muscle fiber CSA and specific atrophy of type IIx/IIb fibers. It will be important to investigate the mechanistic underpinnings of hypoglossal motor neuron death and tongue muscle weakness to eventually provide therapies for age-associated lingual dysfunctions.

Mucosal damage and γ-H2AX formation in the rat urinary bladder induced by aromatic amines with structures similar to o-toluidine and o-anisidine.

Although aromatic amines are widely used as raw materials for dyes, some, such as o-toluidine and o-anisidine, have shown concerning results regarding carcinogenicity in the urinary bladder. We have recently developed a short-term detection method for bladder carcinogens using immunohistochemistry for γ-H2AX, a DNA damage marker. Here, using this method, we evaluated aromatic amines with structures similar to o-toluidine and o-anisidine for bladder mucosal damage and potential carcinogenicity. In total, 17 aromatic amines were orally administered to male F344 rats for 28days, and histopathological examination and γ-H2AX immunostaining of the urinary bladder were performed. Histopathological analysis revealed that seven aromatic amines, including 4-chloro-o-toluidine (4-CT), o-aminoazotoluene, 2-aminobenzyl alcohol (ABA), o-acetotoluidine (o-AT), 3,3'-dimethoxybenzidine, 4-aminoazobenzene (AAB), and 4,4'-methylenedianiline (MDA), induced various bladder lesions, such as hemorrhage, necrosis, and urothelial hyperplasia. The morphological characteristics of mucosal damage induced by these substances were divided into two major types: those resembling o-toluidine and those resembling o-anisidine. Six of these aromatic amines, excluding MDA, also caused significant increases in γ-H2AX formation in the bladder urothelium. Interestingly, 4-CT did not cause mucosal damage or γ-H2AX formation at the lower dose applied in previous carcinogenicity studies. These results showed for the first time that o-AT and ABA, metabolites of o-toluidine, as well as AAB caused damage to the bladder mucosa and suggested that they may be bladder carcinogens. In addition, 4-CT, which was thought to be a noncarcinogen, was found to exhibit bladder toxicity upon exposure to high doses, indicating that this compound may contribute to bladder carcinogenesis.

Maternal dexamethasone exposure does not affect glucose tolerance but alters renal haemodynamics in F1 rats in a sex-dependent manner.

Prenatal programming with dexamethasone increases the risk of the development of hyperglycaemia and insulin resistance, leading to diabetes in adulthood. Dexamethasone also causes a decline in renal glomerular filtration in the adult offspring. Sodium-glucose cotransporter-2 (SGLT2) plays a significant role in regulating blood glucose and renal haemodynamics in diabetic patients. However, the role of SGLT2 in dexamethasone-induced programming and the putative sex-dependent effects on the changes named earlier is unknown. Therefore, this study aimed to investigate the impact of maternal dexamethasone treatment on glucose tolerance, insulin sensitivity, renal perfusion and renal function in adult male and female offspring and the possible contribution of SGLT2 to these changes. Pregnant Sprague Dawley rats (F0 ) were treated with either vehicle or dexamethasone (0.2 mg/kg ip) from gestation Day 15 to 20. F1 males and F1 females were randomly selected from each mother at 4 months of age. There was no change in serum Na+ , Na+ excretion rate, glucose tolerance or insulin sensitivity in F1 male or female rats. However, dexamethasone caused significant glomerular hypertrophy and decreases in CSinistrin and CPAH indicating decreased glomerular filtration rate and renal plasma flow, respectively, in dexamethasone-treated F1 male but not female rats. Dexamethasone did not affect SGLT2 mRNA or protein expression in F1 males or females. We conclude that dexamethasone-mediated prenatal programming of glomerular volume, renal function and haemodynamics is sex-dependent, occurring only in adult male offspring.

Immunotoxicity of N-butylbenzenesulfonamide: impacts on immune function in adult mice and developmentally exposed rats.

N-butylbenzenesulfonamide (NBBS) is a high-production volume plasticizer that is an emerging contaminant of concern for environmental and human health. To understand the risks and health effects of exposure to NBBS, studies were conducted in adult-exposed mice and developmentally exposed rats to evaluate the potential for NBBS to modulate the immune system. Beginning between 8 and 9 weeks of age, dosed feed containing NBBS at concentrations of 0, 313, 625, 1250, 2500, and 5000 ppm was continuously provided to B6C3F1/N female mice for 28 days. Dosed feed was also continuously provided to time-mated Harlan Sprague Dawley (Sprague Dawley SD) rats at concentrations of 0-, 250-, 500-, and 1000-ppm NBBS from gestation day 6 to postnatal day 28 and in F1 rats until 11-14 weeks of age. Functional assessments of innate, humoral, and cell-mediated immunity were conducted in adult female mice and F1 rats following exposure to NBBS. In female mice, NBBS treatment suppressed the antibody-forming cell (AFC) response to SRBC with small increases in T-cell responses and natural killer (NK)-cell activity. In developmentally exposed rats, NBBS treatment-related immune effects were sex dependent. A positive trend in NK-cell activity occurred in male F1 rats while a negative trend occurred in female F1 rats. The AFC response to SRBC was decreased in female F1 rats but not in male F1 rats. These data provide evidence that oral exposure to NBBS has the potential to produce immunomodulatory effects on both innate and adaptive immune responses, and these effects appear to have some dependence on species, sex, and period of exposure (developmental vs adult).

L-Arginine abrogates maternal and pre-pubertal codeine exposure-induced impaired spermatogenesis and sperm quality by modulating the levels of mRNA encoding spermatogenic genes.

Although, codeine has been demonstrated to lower sperm quality; the effects of maternal and prepubertal codeine exposure on male offspring is yet to be reported. In addition, the effect of arginine on codeine-induced decline in sperm quality has not been explored. This study investigated the impact of maternal and prepubertal codeine exposure on spermatogenesis and sperm quality in F1 male Wistar rats to study the effect that codeine may have during recreational use in humans. Also, the effect of arginine supplementation on codeine-induced alteration in spermatogenesis and sperm quality was evaluated. Female rats were treated with either 0.5 ml distilled water or codeine orally for eight weeks, and then mated with male rats (female:male, 2:1). The F1 male offsprings of both cohorts were weaned at 3 weeks old and administered distilled water, codeine, arginine, or codeine with arginine orally for eight weeks. Prepubertal codeine exposure in rats whose dams (female parents) were exposed to codeine delayed puberty and reduced the weight at puberty. Prepubertal codeine exposure exacerbated maternal codeine exposure-induced reduced total and daily spermatid production, sperm count, sperm motility, and normal sperm form, as well as impaired sperm plasma membrane integrity and increased not intact acrosome and damaged sperm DNA integrity. These perturbations were accompanied by a decrease in mRNA levels encoding spermatogenic genes, testicular testosterone and androgen receptor (AR) concentrations, and upregulation of sperm 8-hydroxydeoxyguanosine (8OHdG). Prepubertal arginine supplementation mitigated codeine-induced alterations. This study provides novel experimental evidence that maternal and prepubertal codeine exposure reprogramed spermatogenesis and sperm quality of male FI generation by decreasing mRNA levels encoding spermatogenic genes and AR via oxidative stress-mediated signaling, which was abrogated by prepubertal arginine supplementation.

Transgenerational Effects and Mechanisms of Tributyltin Exposure on Neurodevelopment in the Male Offspring of Rats.

This study aimed to investigate the transgenerational effects of tributyltin exposure on rat neurodevelopment in male offspring and the potential mechanisms. Neonatal female rats were exposed to the environmental level of tributyltin and then mated with nonexposed males after sexual maturity to produce the F1 generation. The F1 generation (with primordial germ cell exposure) was mated with nonexposed males to produce nonexposed offspring (the F2 and F3 generations). Neurodevelopmental indicators and behavior were observed for the F1, F2, and F3 generations during postnatal days 1-25 and 35-56, respectively. We found premature eye-opening and delayed visual positioning in newborn F1 rats and anxiety and cognitive deficits in prepubertal F1 male rats. These neurodevelopmental impacts were also observed in F2 and F3 males. Additionally, F1-F3 males exhibited increased serotonin and dopamine levels and a loose arrangement of neurons in the hippocampus. We also observed a reduction in the expression of genes involved in intercellular adhesion and increased DNA methylation of the Dsc3 promoter in F1-F3 males. We concluded that tributyltin exposure led to transgenerational effects on neurodevelopment via epigenetic reprogramming in male offspring. These findings provide insights into the risks of neurodevelopmental disorders in offspring from parents exposed to tributyltin.

Downregulation of striatal CaV1.3 inhibits the escalation of levodopa-induced dyskinesia in male and female parkinsonian rats of advanced age

In the past 25 years, the prevalence of Parkinson's disease (PD) has nearly doubled. Age remains the primary risk factor for PD and as the global aging population increases this trend is predicted to continue. Even when treated with levodopa, the gold standard dopamine (DA) replacement therapy, individuals with PD frequently develop therapeutic side effects. Levodopa-induced dyskinesia (LID), a common side effect of long-term levodopa use, represents a significant unmet clinical need in the treatment of PD. Previously, in young adult (3-month-old) male parkinsonian rats, we demonstrated that the silencing of CaV1.3 (Cacan1d) L-type voltage-gated calcium channels via striatal delivery of rAAV-CaV1.3-shRNA provides uniform protection against the induction of LID, and significant reduction of established severe LID. With the goal of more closely replicating a clinical demographic, the current study examined the effects of CaV1.3-targeted gene therapy on LID escalation in male and female parkinsonian rats of advanced age (18-month-old at study completion). We tested the hypothesis that silencing aberrant CaV1.3 channel activity in the parkinsonian striatum would prevent moderate to severe dyskinesia with levodopa dose escalation. To test this hypothesis, 15-month-old male and female F344 rats were rendered unilaterally parkinsonian and primed with low-dose (3–4 mg/kg) levodopa. Following the establishment of stable, mild dyskinesias, rats received an intrastriatal injection of either the Cacna1d-specific rAAV-CaV1.3-shRNA vector (CAV-shRNA), or the scramble control rAAV-SCR-shRNA vector (SCR-shRNA). Daily (M-Fr) low-dose levodopa was maintained for 4 weeks during the vector transduction and gene silencing window followed by escalation to 6 mg/kg, then to 12 mg/kg levodopa. SCR-shRNA-shRNA rats showed stable LID expression with low-dose levodopa and the predicted escalation of LID severity with increased levodopa doses. Conversely, complex behavioral responses were observed in aged rats receiving CAV-shRNA, with approximately half of the male and female subjects—therapeutic ‘Responders’—demonstrating protection against LID escalation, while the remaining half—therapeutic ‘Non-Responders’—showed LID escalation similar to SCR-shRNA rats. Post-mortem histological analyses revealed individual variability in the detection of Cacna1d regulation in the DA-depleted striatum of aged rats. However, taken together, male and female therapeutic ‘Responder’ rats receiving CAV-shRNA had significantly less striatal Cacna1d in their vector-injected striatum relative to contralateral striatum than those with SCR-shRNA. The current data suggest that mRNA-level silencing of striatal CaV1.3 channels maintains potency in a clinically relevant in vivo scenario by preventing dose-dependent dyskinesia escalation in rats of advanced age. As compared to the uniform response previously reported in young male rats, there was notable variability between individual aged rats, particularly females, in the current study. Future investigations are needed to derive the sex-specific and age-related mechanisms which underlie variable responses to gene therapy and to elucidate factors which determine the therapeutic efficacy of treatment for PD.

Effect of peripheral cellular senescence on brain aging and cognitive decline.

We examine similar and differential effects of two senolytic treatments, ABT-263 and dasatinib + quercetin (D + Q), in preserving cognition, markers of peripheral senescence, and markers of brain aging thought to underlie cognitive decline. Male F344 rats were treated from 12 to 18 months of age with D + Q, ABT-263, or vehicle, and were compared to young (6months). Both senolytic treatments rescued memory, preserved the blood-brain barrier (BBB) integrity, and prevented the age-related decline in hippocampal N-methyl-D-aspartate receptor (NMDAR) function associated with impaired cognition. Senolytic treatments decreased senescence-associated secretory phenotype (SASP) and inflammatory cytokines/chemokines in the plasma (IL-1β, IP-10, and RANTES), with some markers more responsive to D + Q (TNFα) or ABT-263 (IFNγ, leptin, EGF). ABT-263 was more effective in decreasing senescence genes in the spleen. Both senolytic treatments decreased the expression of immune response and oxidative stress genes and increased the expression of synaptic genes in the dentate gyrus (DG). However, D + Q influenced twice as many genes as ABT-263. Relative to D + Q, the ABT-263 group exhibited increased expression of DG genes linked to cell death and negative regulation of apoptosis and microglial cell activation. Furthermore, D + Q was more effective at decreasing morphological markers of microglial activation. The results indicate that preserved cognition was associated with the removal of peripheral senescent cells, decreasing systemic inflammation that normally drives neuroinflammation, BBB breakdown, and impaired synaptic function. Dissimilarities associated with brain transcription indicate divergence in central mechanisms, possibly due to differential access.

A new 90-day drinking water study of sodium cyanide in rats to further evaluate National Toxicology Program findings and inform risk assessment.

The National Toxicology Program (NTP, 1993) reported male reproductive effects in a sodium cyanide (NaCN) drinking water study. The critical effect, decreased cauda epididymis weights, was used by U.S. Environmental Protection Agency for their hazard characterization and risk assessment of hydrogen cyanide and cyanide salts. To further investigate potential male reproductive effects, we conducted a new 90-day drinking water study. Our study expanded evaluations of testes and thyroid. Male F344 rats received NaCN in drinking water at 0, 0 (water restricted; paired to top dose), 3, 10, 30, 100, and 300 ppm for 13 weeks, followed by 10-weeks recovery. Plasma thiocyanate increased dose-dependently but returned to baseline during recovery. NaCN caused neither effects on survival, body weight, food consumption, hematology, serum chemistry, urinalysis, thyroid hormones, testes or epididymides weights, sperm motility/viability, sperm morphology, or sperm production; nor clinical, ophthalmic, or histopathologic findings. Increased organ weights in thyroid/parathyroid and liver occurred at 300-ppm but were recoverable. No changes occurred in male reproductive organs. Absent adverse effects, the NOAEL was 300 ppm (21.66 mg/kg/day; highest dose tested). Based on organ weight increases at 300 ppm, the NOEL was 100 ppm (7.46 mg/kg/day).

Mechanisms of pulmonary disease in F344 rats after workplace-relevant inhalation exposure to cross-linked water-soluble acrylic acid polymers

BackgroundRecently in Japan, six workers at a chemical plant that manufactures resins developed interstitial lung diseases after being involved in loading and packing cross-linked water-soluble acrylic acid polymers (CWAAPs). The present study focused on assessing lung damage in rats caused by workplace-relevant inhalation exposure to CWAAP and investigated the molecular and cellular mechanisms involved in lung lesion development.MethodsUsing a whole-body inhalation exposure system, male F344 rats were exposed once to 40 or 100 mg/m3 of CWAAP-A for 4 h or to 15 or 40 mg/m3 of CWAAP-A for 4 h per day once per week for 2 months (9 exposures). In a separate set of experiments, male F344 rats were administered 1 mg/kg CWAAP-A or CWAAP-B by intratracheal instillation once every 2 weeks for 2 months (5 doses). Lung tissues, mediastinal lymph nodes, and bronchoalveolar lavage fluid were collected and subjected to biological and histopathological analyses.ResultsA single 4-h exposure to CWAAP-A caused alveolar injury, and repeated exposures resulted in regenerative changes in the alveolar epithelium with activation of TGFβ signaling. During the recovery period after the last exposure, some alveolar lesions were partially healed, but other lesions developed into alveolitis with fibrous thickening of the alveolar septum. Rats administered CWAAP-A by intratracheal instillation developed qualitatively similar pulmonary pathology as rats exposed to CWAAP-A by inhalation. At 2 weeks after intratracheal instillation, rats administered CWAAP-B appeared to have a slightly higher degree of lung lesions compared to rats administered CWAAP-A, however, there was no difference in pulmonary lesions in the CWAAP-A and CWAAP-B exposed rats examined 18 weeks after administration of these materials.ConclusionsThe present study reports our findings on the cellular and molecular mechanisms of pulmonary disease in rats after workplace-relevant inhalation exposure to CWAAP-A. This study also demonstrates that the lung pathogenesis of rats exposed to CWAAP-A by systemic inhalation was qualitatively similar to that of rats administered CWAAP-A by intratracheal instillation.Graphical

Dry Beans Inhibit Azoxymethane-Induced Colon Carcinogenesis in F344 Rats

Epidemiological studies show a low incidence of colon cancer in many Latin American countries where the consumption of dry beans (e.g., pinto) is high. The purpose of this study was to use rats as an animal model to obtain experimental data on the inhibition of colon carcinogenesis by dry beans. Fifty-three 5-wk-old weanling male F344 rats were randomly assigned by weight to the following groups: control (11 rats), casein diet (21 rats), and bean diet (21 rats). Animals fed the casein and bean diets were treated with the carcinogen azoxymethane (AOM) once weekly for 2 wk. Rats in the control group also consumed the casein diet but were not exposed to AOM. All diets were isocaloric. The protein concentration of the diets was adjusted to 18 g/100 g with casein, and the fat concentration was adjusted to 5 g/100 g with corn oil. Rats fed the bean diet had significantly fewer colon adenocarcinomas (P< 0.05) than rats fed the casein diet (5 vs. 22 tumors), and significantly fewer rats fed the bean diet (P< 0.05) had colonic tumors than did casein-fed rats (24 vs. 50%). Tumor multiplicity was also significantly lower for the bean-fed rats, and significantly fewer (P< 0.05) tumors per tumor-bearing rat were observed in bean-fed rats than in casein-fed rats (1.0 ± 0.0 vs. 2.5 ± 0.6). This study demonstrates that dry beans contain anticarcinogenic compounds capable of inhibiting AOM-induced colon cancer in rats. However, the specific anticarcinogenic components within dry beans have not been identified, and it is unclear whether dietary fiber, phytochemicals or other components within dry beans are primarily responsible for the anticarcinogenic properties of beans.

Intrauterine exposure to di(2-ethylhexyl) phthalate (DEHP) disrupts the function of the hypothalamus-pituitary-thyroid axis of the F1 rats during adult life.

DEHP is an endocrine disruptor widely used in the production of malleable plastics. DEHP exposure was associated with altered hypothalamic-pituitary-thyroid (HPT) axis function. Although previous studies reported deleterious effects of DEHP exposure during the intrauterine period, few studies have evaluated the direct effects triggered by this endocrine disruptor on the offspring animals' thyroid function. This study aimed to investigate the impact of intrauterine exposure to DEHP on the HPT axis function programming of the offspring animals during adulthood. Pregnant Wistar rats were orally treated with corn oil or corn oil supplemented with DEHP (0.48 or 4.8 mg/kg/day) throughout the gestational period. The offspring rats were euthanized on the 90th postnatal day. Hypothalamus, pituitary, thyroid, and liver were collected to analyze gene expression and protein content through qPCR and Western Blot. Blood was collected to determine TSH and thyroid hormone levels through fluorometric or chemiluminescence immunoassays. In the adult F1 female rats, the highest dose of DEHP decreased TSH serum levels. In the thyroid, DEHP reduced the gene expression and/or protein content of NIS, TSHR, TG, TPO, MCT8, NKX2.1, PAX8, and FOXE1. These data are consistent with the reduction in T4 serum levels of the F1 DEHP-exposed female rats. In the liver, DEHP exposure increased the mRNA expression of Dio1 and Ttr, while the highest dose of DEHP reduced the mRNA expression of Ugt1a1 and Ugt1a6. Conversely, in the F1 male adult rats, TSHB expression and TSH serum levels were increased in DEHP-exposed animals. In the thyroid, except for the reduced protein content of TSHR, none of the evaluated genes/proteins were altered by DEHP. TH serum levels were not changed in the DEHP-exposed F1 male rats compared to the control group. Additionally, there were no significant alterations in the expression of hepatic enzymes in these animals. Our results demonstrated, for the first time, that intrauterine exposure to DEHP disrupts the HPT axis function in male and female offspring rats and strongly suggest that DEHP exposure increases the susceptibility of the offspring animals to develop thyroid dysfunctions during adulthood.

Multi-organ carcinogenicity by inhalation exposure to 2-Bromopropane in rats.

The purpose of this study was to investigate the carcinogenicity of 2-bromopropane (2-BP) in rats. Male and female F344 rats were exposed by whole body inhalation to 2-BP vapor at concentrations of 0, 67, 200, and 600 ppm for 6h/day, 5 days/week for 2 years. All rats of both sexes exposed to 600 ppm died or became moribund within 85 weeks. Death/moribunditywas caused by 2-BP induced tumors. In males, significantly increased tumors were malignant Zymbal's gland tumors; sebaceous adenoma and basal cell carcinoma of the skin/appendage; adenocarcinoma of the small/large intestine; follicular cell adenoma of the thyroid; fibroma of the subcutis, and malignant lymphoma of the lymph node. In addition, an increased trend in tumor incidence was found in the preputial gland, lung, forestomach, pancreas islet, brain, and spleen. In females, significantly increased tumors were adenocarcinoma and fibroadenoma of the mammary gland, squamous cell papilloma of the vagina, and large granular lymphocytic leukemia of the spleen. In addition, an increased trend in tumor incidence was found in Zymbal's gland, the clitoral gland, skin, large intestine, pancreas islet, uterus, and subcutis. Particularly, malignant Zymbal's gland tumors were induced even in males exposed to the lowest concentration, 67 ppm. Two-year inhalation exposure to 2-BP resulted in multi-organ carcinogenicity in rats. Based on sufficient evidence of carcinogenicity in this study, 2-BP has the potential to be a human carcinogen.

Early detection of hepatocarcinogens in rats by immunohistochemistry of γ-H2AX.

We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of γ-H2AX, a well-known marker of DNA damage. To investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens, we examined γ-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitrosodiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3'-dimethylbenzidine dihydrochloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for γ-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in γ-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Brip1, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with γ-H2AX formation. These results suggest that γ-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether γ-H2AX immunostaining should be combined with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.

Incorporation of a recirculating mobile lipid pool description into the cyclic volatile siloxane (cVMS) PBPK model captures terminal clearance of D4 after repeated inhalation exposure in F344 and SD Rats

The most recent version of the octamethylcyclotetrasiloxane (D4) physiologically based pharmacokinetic (model) was developed using the available kinetic studies in male and female F344 rats. Additional data, which had not been included in the D4 model development, allowed for a more detailed assessment of the loss of D4 following long-term exposure in both SD and F344 rats. This new data demonstrated a deficiency in the published PBPK model predictions of terminal concentrations of D4 in plasma and fat 14 days after the end of exposures for 28-days, 6 h/day, where the model predictions were an order of magnitude lower than the data. To capture this time-point without altering the end-of-exposure peak concentrations in blood and fat required conversion of the one-way (liver to fat) mobile lipoprotein pool (MLP) into a bi-directional pool between liver and fat. Simulation of the D4 pharmacokinetics in the SD rat, as opposed to the F344 rat, also required a reduction of both fold induction of liver metabolism (KMAX: 5- to 2-fold) and maximal rate of metabolism (VMAXC: 5.0–1.54 mg/kg0.75). The revised MLP description was extended to the human D4 model using a parallelogram approach between rat and human MLP parameters to establish the parameters for the current model in the absence of similar long-term clearance data in the human. The revised human D4 model provided good fits to the human inhalation and dermal exposure studies while not appreciably altering cross-species dose metrics based on the free concentration of D4 in blood.

An evaluation of the rat intestinal monoamine biogeography days following exposure to acute stress.

Stress-induced abnormalities in gut monoamine levels (e.g., serotonin, dopamine, norepinephrine) have been linked to gastrointestinal (GI) dysfunction, as well as the worsening of symptoms in GI disorders. However, the influence of stress on changes across the entire intestinal monoamine biogeography has not been well-characterized, especially in the days following stress exposure. Therefore, the aim of this study was to comprehensively assess changes to monoamine neurochemical signatures across the entire rat intestinal tract days after exposure to an acute stressor. To the end, adult male F344 rats were subjected to an episode of unpredictable tail shocks (acute stress) or left undisturbed. Forty-eight hours later rats were euthanized either following a 12h period of fasting or 30min of food access to evaluate neurochemical profiles during the peri- and early postprandial periods. Monoamine-related neurochemicals were measured via UHPLC in regions of the small intestine (duodenum, jejunum, ileum), large intestine (cecum, proximal colon, distal colon), cecal contents, fecal contents, and liver. The results suggest a relatively wide-spread increase in measures of serotonin activity across intestinal regions can be observed 48h after exposure to acute stress, however some evidence was found supporting localized differences in serotonin metabolization. Moreover, acute stress exposure reduced catecholamine-related neurochemical concentrations most notably in the ileum, and to a lesser extent in the cecal contents. Next, stress-related fecal serotonin concentrations were consistent with intestinal profiles. However, fecal dopamine was elevated in association with stress, which did not parallel findings in any other intestinal area. Finally, stress exposure and the food access period together only had minor effects on intestinal monoamine profiles. Taken together, these data suggest nuanced differences in monoaminergic profiles exist across intestinal regions the days following exposure to an acute stressor, highlighting the importance of assessments that consider the entire intestinal tract biogeography when investigating stress-related biological outcomes that may be relevant to GI pathophysiology.

Abstract IA005: Discovery and development of LFA-9 for CRC prevention

Abstract Colorectal Cancer (CRC) is a major public health issue with an estimated 980,000 deaths annually, worldwide. Current trends show an increase of CRC incidence and mortality worldwide. Colonic adenomas are very common in ages 50 and over. Though, nonsteroidal antiinflammatory agents like Aspirin, Naproxen and COX-2 inhibitor and Celecoxib are useful to prevent the polyp progression and reducing CRC burden, continuous/chronic usage of these drugs are limited by GI toxicity and unwanted side effects. Thus, the rationale to establish safer anti-inflammatory agents for CRC prevention is important. Mechanistic studies suggest that up-regulation of 15-PGDH, sparing COX-1/2 and prostaglandin (PG)I2 synthase and/or selectively targeting mPGES-1 and 5-LOX would reduce the cardiovascular side effects and may improve the chemopreventive efficacy. There are limited to no studies on targeting mPGES-1/5LOX and it is one of the unexplored areas in colorectal cancer chemoprevention towards safer drug development. To design and develop selective inhibitors of dual mPGES-1/5-LOX, we used in-silico small molecular docking simulation approaches, and identified LFA-9 as a novel duel mPGES-1/5-LOX inhibitor among &amp;gt;35 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized as ex-vivo to assess pharmacodynamic inhibitory effects of LFA-9 on mPGES-1 and 5-LOX by Radio-HPLC. In a series of animal experiments, we established the dose-range of toxicity and optimal doses of LFA-9. Potential preventive efficacy of LFA-9 was assessed AOM-rat colon carcinogenesis (Aberrant Crypt Foci, (ACF) as surrogate marker in male F344 rats and intestinal tumor inhibition in APCMin/+ mice. Furthermore, we have shown the efficacy of LFA-9 administered in the diet at the adenoma stage significantly suppressed colonic adenocarcinoma formation in AOM-induced colon cancer (sporadic CRC model) and PIRC rat model (FAP model). Overall, the above results show that LFA-9, a novel dual mPGES-1/5-LOX inhibitor, is a safer agent and has potential for prevention of CRC in high-risk patients. {This work was supported by NCI-PREVENT Cancer Program, R01 CA213987 and VA Merit Awards} Citation Format: Chinthalapally V. Rao. Discovery and development of LFA-9 for CRC prevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA005.

Platelet-Derived Microvesicles Promote Selective Cell Viability in Hepatocellular Carcinoma: Differences in Properties between Normal Platelets and Tumor Educated Platelets

Tumor cells educate platelets by modulating their RNA profiles and phenotypes. These platelets are termed "tumor educated platelets: TEPs", and this concept is widely accepted. We have previously reported that platelets are activated in microvessels within liver tumors, leading to the progression of hepatocellular carcinoma (HCC). When platelets are activated, platelet-derived microvesicles (PDMV) containing platelet-derived nucleic acids and proteins are released. The contents in these are known to vary depending on the various pathological conditions of the host. It is generally believed that when cells take up extracellular vesicles, they are affected by the action of the encapsulated substances. However, it is still unclear what biological properties of PDMV are released by TEPs and how they are involved in carcinogenesis. We hypothesized that the release of PDMV by activated platelets in liver tissue and the uptake of its contents by normal hepatocytes and HCC cells may be involved in the progression of hepatocarcinogenesis, and we analyzed their role in carcinogenesis. The rat HCC model was prepared according to the Solt-Farber protocol. Briefly, male F344 rats were injected intraperitoneally with diethylnitrosamine at 4 weeks of age, then fed a powdered diet containing 2-acetylaminofluorene to induce liver damage, followed by partial hepatectomy. The rats were then kept under normal conditions for more than one month. Abdominal ultrasonography was performed on rats, and those with obvious tumors with a diameter of 2 mm or more were used for the experiments. Peripheral blood was collected from the tail veins of normal rats or HCC model rats, and platelets were isolated. The platelets were labeled with the fluorescent substance CMRA, activated in vitro with thrombin, and the PDMV in the supernatant was collected by polyethylene glycol precipitation and used as CMRA-labeled PDMV in the experiments. When PDMV was added to primary cultured rat hepatocytes and cultured for 24 hours, fluorescence was observed in the hepatocytes. The fluorescence was also observed in hepatocytes in the portal region of liver tissue collected 24 hours after injection of CMRA-labeled PDMV from the mesenteric vein of rats. These indicate that PDMV released into the liver tissue is taken up by hepatocytes. Peripheral blood platelets from normal rats or HCC model rats were isolated and PDMV was purified from each (N-PDMV and HCC-PDMV). These PDMVs were added to cultures of normal rat hepatocytes and hepatocellular carcinoma cell lines to analyze their effects on cell survival. N-PDMV did not affect the viability of normal hepatocytes, but mildly enhanced the survival of HCC cells. On the other hand, HCC-PDMV inhibited the viability of normal hepatocytes, but promoted viability of HCC cells more strongly than N-PDMV. These results indicate that the contents of PDMV differ between normal and HCC rats, and in particular that PDMV from rats with HCC is taken up by normal and HCC cells and is associated with selective survival of HCC cells.