The Natural History of Acute Radiation-induced H-ARS and Concomitant Multi-organ Injury in the Non-human Primate: The MCART Experience.

ABSTRACT: The dose response relationship and corresponding values for mid-lethal dose and slope are used to define the dose- and time-dependent parameters of the hematopoietic acute radiation syndrome. The characteristic time course of mortality, morbidity, and secondary endpoints are well defined. The concomitant comorbidities, potential mortality, and other multi-organ injuries that are similarly dose- and time-dependent are less defined. Determination of the natural history or pathophysiology associated with the lethal hematopoietic acute radiation syndrome is a significant gap in knowledge, especially when considered in the context of a nuclear weapon scenario. In this regard, the exposure is likely ill-defined, heterogenous, and nonuniform. These conditions forecast sparing of bone marrow and increased survival from the acute radiation syndrome consequent to threshold doses for the delayed effects of acute radiation exposure due to marrow sparing, medical management, and use of approved medical countermeasures. The intent herein is to provide a composite natural history of the pathophysiology concomitant with the evolution of the potentially lethal hematopoietic acute radiation syndrome derived from studies that focused on total body irradiation and partial body irradiation with bone marrow sparing. The marked differential in estimated LD50/60 from 7.5 Gy to 10.88 Gy for the total body irradiation and partial body irradiation with 5% bone marrow sparing models, respectively, provided a clear distinction between the attendant multiple organ injury and natural history of the two models that included medical management. Total body irradiation was focused on equivalent LD50/60 exposures. The 10 Gy and 11 Gy partial body with 5% bone marrow sparing exposures bracketed the LD50/60 (10.88 Gy). The incidence, progression, and duration of multiple organ injury was described for each exposure protocol within the hematopoietic acute radiation syndrome. The higher threshold doses for the partial body irradiation with bone marrow sparing protocol induced a marked degree of multiple organ injury to include lethal gastrointestinal acute radiation syndrome, prolonged crypt loss and mucosal damage, immune suppression, acute kidney injury, body weight loss, and added clinical comorbidities that defined a complex timeline of organ injury through the acute hematopoietic acute radiation syndrome. The natural history of the acute radiation syndrome presents a 60-d time segment of multi-organ sequelae that is concomitant with the latent period or time to onset of the evolving multi-organ injury of the delayed effects of acute radiation exposure.

Scientific and Regulatory Policy Committee Best Practices: Documentation of Sexual Maturity by Microscopic Evaluation in Nonclinical Safety Studies.

The sexual maturity status of animals in nonclinical safety studies can have a significant impact on the microscopic assessment of the reproductive system, the interpretation of potential test article-related findings, and ultimately the assessment of potential risk to humans. However, the assessment and documentation of sexual maturity for animals in nonclinical safety studies is not conducted in a consistent manner across the pharmaceutical and chemical industries. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology convened an international working group of pathologists and nonclinical safety scientists with expertise in the reproductive system, pathology nomenclature, and Standard for Exchange of Nonclinical Data requirements. This article describes the best practices for documentation of the light microscopic assessment of sexual maturity in males and females for both rodent and nonrodent nonclinical safety studies. In addition, a review of the microscopic features of the immature, peripubertal, and mature male and female reproductive system and general considerations for study types and reporting are provided to aid the study pathologist tasked with documentation of sexual maturity.

Acute Radiation-induced Lung Injury in the Non-human Primate: A Review and Comparison of Mortality and Co-morbidities Using Models of Partial-body Irradiation with Marginal Bone Marrow Sparing and Whole Thorax Lung Irradiation.

The nonhuman primate, rhesus macaque, is a relevant animal model that has been used to determine the efficacy of medical countermeasures to mitigate major signs of morbidity and mortality of radiation-induced lung injury. Herein, a literature review of published studies showing the evolution of lethal lung injury characteristic of the delayed effects of acute radiation exposure between the two significantly different exposure protocols, whole thorax lung irradiation and partial-body irradiation with bone marrow sparing in the nonhuman primate, is provided. The selection of published data was made from the open literature. The primary studies conducted at two research sites benefitted from the similarity of major variables; namely, both sites used rhesus macaques of approximate age and body weight and radiation exposure by LINAC-derived 6 MV photons at dose rates of 0.80 Gy min and 1.00 Gy min delivered to the midline tissue via bilateral, anterior/posterior, posterior/anterior geometry. An advantage relative to sex difference resulted from the use of male and female macaques by the Maryland and the Washington sites, respectively. Subject-based medical management was used for all macaques. The primary studies (6) provided adequate data to establish dose response relationships within 180 d for the radiation-induced lung injury consequent to whole thorax lung irradiation (male vs. female) and partial-body irradiation with bone marrow sparing exposure protocols (male). The dose response relationships established by probit analyses vs. linear dose relationships were characterized by two main parameters or dependent variables, a slope and LD50/180. Respective LD50/180 values for the primary studies that used whole thorax lung irradiation for respective male and female nonhuman primates were 10.24 Gy [9.87, 10.52] (n = 76, male) and 10.28 Gy [9.68, 10.92] (n = 40, female) at two different research sites. The respective slopes were steep at 1.73 [0.841, 2.604] and 1.15 [0.65, 1.65] probits per linear dose. The LD50/180 value and slope derived from the dose response relationships for the partial-body irradiation with bone marrow sparing exposure was 9.94 Gy [9.35, 10.29] (n = 87) and 1.21 [0.70, 1.73] probits per linear dose. A secondary study (1) provided data on limited control cohort of nonhuman primates exposed to whole thorax lung irradiation. The data supported the incidence of clinical, radiographic, and histological indices of the dose-dependent lung injury in the nonhuman primates. Tertiary studies (6) provided data derived from collaboration with the noted primary and secondary studies on control cohorts of nonhuman primates exposed to whole thorax lung irradiation and partial-body irradiation with bone marrow sparing exposure. These studies provided a summary of histological evidence of fibrosis, inflammation and reactive/proliferative changes in pneumonocytes characteristic of lung injury and data on biomarkers for radiation-induced lung injury based on matrix-assisted laser desorption ionization-mass spectrometry imaging and gene expression approaches. The available database in young rhesus macaques exposed to whole thorax lung irradiation or partial-body irradiation with bone marrow sparing using 6 MV LINAC-derived radiation with medical management showed that the dose response relationships were equivalent relative to the primary endpoint all-cause mortality. Additionally, the latency, incidence, severity, and progression of the clinical, radiographic, and histological indices of lung injury were comparable. However, the differences between the exposure protocols are remarkable relative to the demonstrated time course between the multiple organ injury of the acute radiation syndrome and that of the delayed effects of acute radiation exposure, respectively.

Lack of Cellular Inflammation in a Non-human Primate Model of Radiation Nephropathy.

Inflammation is commonly cited as a mechanism of delayed effects of acute radiation exposure (DEARE). Confirmation of its presence could provide significant insight to targeted use of treatments or mitigators of DEARE. We sought to quantify the presence of cellular inflammation in kidneys of non-human primates that developed acute and chronic kidney injury after a partial body irradiation exposure. We show herein that cellular inflammation is not found as a component of either acute or chronic kidney injury. Other mechanistic pathways of injury must be sought.

MALDI-MSI spatially maps N-glycan alterations to histologically distinct pulmonary pathologies following irradiation.

Radiation-induced lung injury is a highly complex combination of pathological alterations that develop over time and severity of disease development is dose-dependent. Following exposures to lethal doses of irradiation, morbidity and mortality can occur due to a combination of edema, pneumonitis and fibrosis. Protein glycosylation has essential roles in a plethora of biological and immunological processes. Alterations in glycosylation profiles have been detected in diseases ranging from infection, inflammation and cancer. We utilized mass spectrometry imaging to spatially map N-glycans to distinct pathological alterations during the clinically latent period and at 180 days post-exposure to irradiation. Results identified alterations in a number of high mannose, hybrid and complex N-glycans that were localized to regions of mucus and alveolar-bronchiolar hyperplasia, proliferations of type 2 epithelial cells, accumulations of macrophages, edema and fibrosis. The glycosylation profiles indicate most alterations occur prior to the onset of clinical symptoms as a result of pathological manifestations. Alterations in five N-glycans were identified as a function of time post-exposure. Understanding the functional roles N-glycans play in the development of these pathologies, particularly in the accumulation of macrophages and their phenotype, may lead to new therapeutic avenues for the treatment of radiation-induced lung injury.

A Pathology Review of the Lower Gastrointestinal Tract in Relation to Ulcerative Colitis in Rats and Cynomolgus Macaques Treated With Ammonium Perfluorooctanoate.

Among many short-term, subchronic, and chronic toxicology studies with ammonium perfluorooctanoate (PFOA), the gastrointestinal tract has not been identified as a target organ for PFOA-related toxicity in laboratory animals where the corresponding serum PFOA concentrations typically approach several orders of magnitude higher than the general human population. These lack of gastrointestinal tract-related findings were in direct contrast to an epidemiological observation where a positive trend was observed for ulcerative colitis, an idiopathic chronic inflammatory condition of the gut, in a Mid-Ohio River community whose drinking water contained higher levels of PFOA. This study was conducted to perform a histological reevaluation of large intestine sections in laboratory animals from 2 long-term toxicological studies: one was with Sprague Dawley rats that received ammonium PFOA in their diet for 2 years and the other one was with cynomolgus macaques that received daily capsules of ammonium PFOA for 6 months. In both studies, there was a lack of histological evidence of treatment-related inflammatory lesions that was suggestive of the occurrence of ulcerative colitis in these laboratory animals even under the most rigorous treatment schedules. These findings do not offer support for the biological plausibility of the epidemiological associations reported.

Lung and Heart Injury in a Nonhuman Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing: Histopathological Evidence of Lung and Heart Injury.

Male rhesus macaques were subjected to partial-body irradiation at 10, 11, or 12 Gy with 5% bone marrow protection. Animals were euthanized when dictated by prospectively determined clinical parameters or at approximately 180 d following irradiation. Histological sections of lung and heart were stained with hematoxylin and eosin as well as a battery of histochemical and immunohistochemical stains. Histopathological alterations in the lung were centered on fibrosis, inflammation, and reactive/proliferative changes in pneumocytes. These changes were noted in animals necropsied after approximately 85-100 d postirradiation and extending through the observation period. Interstitial and pleural fibrosis demonstrated by Masson's trichrome staining were associated with increased alpha smooth muscle actin and collagen 1 immunohistochemical staining. Areas of interstitial fibrosis had reduced microvascular density with CD31 immunohistochemical staining. Accumulations of CD163- and CD206-positive alveolar macrophages were present in areas of interstitial fibrosis. Unidentified cells termed "myxoid" cells in alveolar walls had histochemical and immunohistochemical staining characteristics of epithelial-, endothelial-, or pericyte-mesenchymal transition states that were developing myofibroblast features. Distinctive focal or multifocal alveolar-bronchiolar hyperplasia had microscopic features of preneoplastic proliferation. Delayed radiation-associated changes in the heart consisted primarily of myocardial fibrosis, with rare histological evidence of myofiber degeneration.

Characterizing the Natural History of Acute Radiation Syndrome of the Gastrointestinal Tract: Combining High Mass and Spatial Resolution Using MALDI-FTICR-MSI.

The acute radiation syndrome of the gastrointestinal tract has been histologically characterized, but the molecular and functional mechanisms that lead to these cellular alterations remain enigmatic. Mass spectrometry imaging is the only technique that enables the simultaneous detection and cellular or regional localization of hundreds of biomolecules in a single experiment. This current study utilized matrix-assisted laser desorption/ionization mass spectrometry imaging for the molecular characterization of the first natural history study of gastrointestinal acute radiation syndrome in the nonhuman primate. Jejunum samples were collected at days 4, 8, 11, 15, and 21 following 12-Gy partial-body irradiation with 2.5% bone marrow sparing. Mass spectrometry imaging investigations identified alterations in lipid species that further understanding of the functional alterations that occur over time in the different cellular regions of the jejunum following exposure to high doses of irradiation. Alterations in phosphatidylinositol species informed on dysfunctional epithelial cell differentiation and maturation. Differences in glycosphingolipids of the villi epithelium that would influence the absorptive capacity and functional structure of the brush border membrane were detected. Dichotomous alterations in cardiolipins indicated altered structural and functional integrity of mitochondria. Phosphatidylglycerol species, known regulators of toll-like receptors, were detected and localized to regions in the lamina propria that contained distinct immune cell populations. These results provide molecular insight that can inform on injury mechanism in a nonhuman primate model of the acute radiation syndrome of the gastrointestinal tract. Findings may contribute to the identification of therapeutic targets and the development of new medical countermeasures.

Radiation Nephropathy in a Nonhuman Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing-Part 1: Acute and Chronic Kidney Injury and the Influence of Neupogen.

Acute and chronic kidney injury may occur after accidental prompt radiation exposures. We have modeled their occurrence in a nonhuman primate model. Subjects who are exposed to more than 5-Gy prompt irradiation are apt to show blood cell cytopenias and be treated with granulocyte colony-stimulating factors such as Neupogen® or Neulasta® to mitigate the hematologic injury of the acute radiation syndrome. Neupogen or Neulasta are now approved by the US Food and Drug Administration for this indication. This will significantly increase the number of survivors of acute radiation exposures who will be at risk for delayed effects of radiation exposure, which includes acute and chronic kidney injury. The primary objectives of the present two companion manuscripts were to assess natural history of delayed radiation-induced renal injury in a nonhuman primate model of acute, high-dose, partial-body irradiation with 5% bone marrow sparing to include the clinical and histopathological evidence and the effect of Neupogen administration on morbidity and mortality. In this study, 88 nonhuman primates underwent 10- or 11-Gy partial-body irradiation with 5% bone marrow sparing, of which 36 were treated with Neupogen within 1, 3, or 5 d postirradiation. All animals were followed up to 180 d after irradiation. Renal function and histology end points showed early acute and later chronic kidney injury. These end points were not affected by use of Neupogen. We conclude that use of Neupogen to mitigate against the hematopoietic acute radiation syndrome has no impact on acute or chronic kidney injury.

Radiation Nephropathy in a Nonhuman Primate Model of Partial-Body Irradiation With Minimal Bone Marrow Sparing-Part 2: Histopathology, Mediators, and Mechanisms.

Male rhesus macaques were subjected to partial-body irradiation at 10, 11, or 12 Gy with 5% bone marrow protection. Animals were euthanized when dictated by prospectively determined clinical parameters or at approximately 180 d following irradiation. Histological sections of kidney were stained with hematoxylin and eosin as well as a battery of histochemical and immunohistochemical stains. Histopathological alterations were centered on glomerular changes and fibrosis of glomeruli and the interstitial compartment. These changes were first noted in animals necropsied approximately 100 d postirradiation and continued in animals necropsied through the observation period. Glomerular changes included congestion, thrombosis, erythrocyte degeneration, capillary tuft dilation, fibrin deposition, altered quantity and dispersion pattern of von Willebrand factor, increased mesangial matrix, and mesangial deposits of material that stained positively with periodic acid-Schiff staining. Areas of interstitial and glomerular fibrosis, as demonstrated by Masson's trichrome staining, were topographically associated with increased immunohistochemical staining for connective tissue growth factor, alpha smooth muscle actin, and collagen 1, but there was little staining for transforming growth factor beta. Fibrotic glomeruli had reduced microvascularity as demonstrated by reduced CD31 immunohistochemical staining. Vascular congestion was commonly noted in the region of the corticomedullary junction, and proteinaceous casts were commonly noted in cortical and medullary tubules. Longitudinal analysis of histopathological alterations provided evidence defining the latency, severity, and progression of delayed radiation-induced kidney injury.

The Time Course of Radiation-induced Lung Injury in a Nonhuman Primate Model of Partial-body Irradiation With Minimal Bone Marrow Sparing: Clinical and Radiographic Evidence and the Effect of Neupogen Administration.

The primary objectives of two companion manuscripts were to assess the natural history of delayed radiation-induced lung injury in a nonhuman primate model of acute high-dose, partial-body irradiation with 5% bone marrow sparing, to include the clinical, radiographic, and histopathological evidence and the effect of Neupogen administration on the morbidity and mortality. Nonhuman primates were exposed to 10.0 or 11.0 Gy with 6 MV linac-derived photons at approximately 0.80 Gy min. All nonhuman primates received subject-based, medical management. Subsets of nonhuman primates were administered Neupogen (10 µg kg) starting on day 1, day 3, or day 5 until recovery (absolute neutrophil count ≥ 1,000 cells µL for three consecutive days). Mortality due to multiple organ injury at 180 d study duration: Mortality at 180 d post either 10.0 Gy or 11.0 Gy was the consequence of concurrent injury due to the acute radiation syndrome (gastrointestinal and hematological) and delayed radiation-induced lung injury. The 180-d all-cause mortality observed in the control cohorts at 10.0 Gy (53%) or 11.0 Gy (86%) did not vary from cohorts that received Neupogen at any administration schedule. Mortality ranged from 43-50% (10 Gy) to 75-100% (11.0 Gy) in the Neupogen-treated cohorts. The study, however, was not powered to detect statistical significant differences between mortality in the control and Neupogen-treated cohorts. Clinical and radiographic evidence of radiation-induced lung injury: The mean nonsedated respiratory rate in the control cohorts exposed to 10 or 11 Gy increased from a baseline value of 37 breaths min to >60 breaths min within 103 d and 94 d postexposure, and the incidence of nonsedated respiratory rate > 80 breaths min was 50% and 70%, respectively. The mean duration of latency to development of clinical pneumonitis and/or fibrosis (nonsedated respiratory rate > 80 breaths min) was not significantly different between the 10.0-Gy or 11.0 Gy-cohorts (range 100-107 d). Neupogen (granulocyte colony-stimulating factor) administration had no apparent effect of the latency, incidence, or severity of nonsedated respiratory rate within either radiation dose or administration schedule. Computed tomography scans were obtained and images were analyzed for evidence of lung injury, e.g., pneumonitis and/or fibrosis, pleural and pericardial effusion. A quantitative, semiautomated method was developed based on differences in radiodensity (Hounsfield units) and lung morphology to extract the volume of pneumonitis/fibrosis and pleural effusion as indexed against total lung at each time point obtained. At both irradiation doses, 100% of the nonhuman primates surviving acute radiation syndrome manifested radiographic evidence of radiation-induced lung injury as pneumonitis and/or fibrosis. There was no apparent effect of Neupogen administration on the latency, incidence, severity, or progression of pneumonitis/fibrosis:total lung volume or pleural effusion:total lung volume at either exposure. A comparative review of the data illustrated the concomitant time course of increased mortality, nonsedated respiratory rate, and pneumonitis/fibrosis:total lung volume and pleural effusion:total lung volume consequent to 10.0-Gy or 11.0-Gy partial-body irradiation with 5% bone marrow sparing. All key parameters proceeded from a latent period of approximately 60 d followed by an increase in all three indices of clinical and radiographic evidence of radiation-induced lung injury within the next 60 d to 120 d postexposure. The subsequent time course and longitudinal analysis was influenced by the persistent progression of radiation-induced lung injury, administration of dexamethasone, and loss of nonhuman primates due to lethality. Companion paper: Lung and Heart Injury in a Nonhuman Primate Model of Partial-body Irradiation With Minimal Bone Marrow Sparing: Histopathological Evidence of Lung and Heart Injury (Parker et al. 2019): Note that the computed tomography-based radiodensity data do not permit differentiation of pneumonitis and fibrosis. The companion paper employed Masson's trichrome, collagen 1, and selected staining to identify the key time and incidence parameters relative to excessive collagen deposition indicative of fibrosis and associated histopathology in the lung. This histological database provided valuable longitudinal analysis in support of the clinical and radiographic evidence associated with the time course of radiation-induced lung injury.

The Gastrointestinal Subsyndrome of the Acute Radiation Syndrome in Rhesus Macaques: A Systematic Review of the Lethal Dose-response Relationship With and Without Medical Management.

Well-characterized animal models that mimic the human response to potentially lethal doses of radiation are required to assess the efficacy of medical countermeasures under the criteria of the US Food and Drug Administration's Animal Rule. Development of a model for the gastrointestinal acute radiation syndrome requires knowledge of the radiation dose-response relationship and time course of mortality and morbidity across the acute and prolonged gastrointestinal radiation syndrome. The nonhuman primate, rhesus macaque, is a relevant animal model that has been used to determine the efficacy of medical countermeasures to mitigate major signs of morbidity and mortality relative to the hematopoietic acute radiation syndrome, gastrointestinal acute radiation syndrome, and lung injury. It can be used to assess the natural history of gastrointestinal damage, concurrent multiple organ injury, and aspects of the mechanism of action for acute radiation exposure and treatment. A systematic review of relevant studies that determined the dose-response relationship for the gastrointestinal acute and prolonged radiation syndrome in the rhesus macaque relative to radiation dose, quality, dose rate, exposure uniformity, and use of medical management has never been performed.

Histopathological Features of the Development of Intestine and Mesenteric Lymph Node Injury in a Nonhuman Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing.

Male rhesus macaques were subjected to partial-body irradiation at 10, 11, or 12 Gy with 5% bone marrow protection. Animals were euthanized when dictated by prospectively determined clinical parameters or at approximately 180 d following irradiation. Histological sections of jejunum, colon, and mesenteric lymph node were stained with hematoxylin and eosin as well as a battery of histochemical and immunohistochemical stains. The immediate postirradiation histopathological alterations in the jejunum and colon were based primarily on injury to rapidly proliferating crypt epithelial cells, though there was evidence of additional radiation-induced fibrogenic responses. There was substantial resolution of the radiation-related mucosal injury through the observation period, but microscopically visible defects in mucosal structure persisted to the end of the observation period. In the later stages of the observation period, the jejunum and colon had overt fibrosis that was most commonly located in the submucosa and serosa, with less microscopically discernible involvement of the mucosa. Mesenteric lymph nodes had an immediate postirradiation reduction in cellularity due to the known effects of irradiation on lymphoid cell populations. In later stages of the observation period the lymph nodes also developed fibrotic changes, possibly related to transmigration of immunomodulatory cells and/or signaling molecules from the radiation-damaged intestine.

Reproductive and developmental toxicity of potassium perfluorohexanesulfonate in CD-1 mice.

Potassium perfluorohexanesulfonate (K+PFHxS) was evaluated for reproductive/developmental toxicity in CD-1 mice. Up to 3 mg/kg-d K+PFHxS was administered (n = 30/sex/group) before mating, for at least 42 days in F0 males, and for F0 females, through gestation and lactation. F1 pups were directly dosed with K+PFHxS for 14 days after weaning. There was an equivocal decrease in live litter size at 1 and 3 mg/kg-d, but the pup-born-to-implant ratio was unaffected. Adaptive hepatocellular hypertrophy was observed, and in 3 mg/kg-d F0 males, it was accompanied by concomitant decreased serum cholesterol and increased alkaline phosphatase. There were no other toxicologically significant findings on reproductive parameters, hematology/clinical pathology/TSH, neurobehavioral effects, or histopathology. There were no treatment-related effects on postnatal survival, development, or onset of preputial separation or vaginal opening in F1 mice. Consistent with previous studies, our data suggest that the potency of PFHxS is much lower than PFOS in rodents.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 2 of 2).

To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 1 of 2).

Spontaneous rodent progressive cardiomyopathy (PCM) in the Sprague Dawley rat may confound identification and/or interpretation of potential test article (TA)-related cardiotoxicity. Pathologists apply diagnostic term(s) and thresholds for diagnosing and assigning severity grades for PCM and/or PCM-like (PCM/like) lesions consistently within a study, which is necessary to identify and interpret TA-related findings. Due to differences in training and/or experiences, diagnostic terms and thresholds may vary between pathologists. Harmonized terminology and thresholds across studies will generate better historical control data, will likely enhance interpretation of study data, and may further enhance our understanding of the spontaneous change. An assessment of the diagnostic approaches of a group of 37 pathologists identified an approach that is relatively easily applied; and if adopted, it could enhance diagnostic consistency across studies. This approach uses the single "slash" term "necrosis/inflammatory cell infiltrate (NICI)" as the diagnosis for the spectrum of lesions seen in younger rats, uses no threshold for diagnosis (e.g., diagnose all lesions clearly identifiable as PCM/like), and uses aggregate lesion size of approximately ≥45% of the field of view (FOV) using a 10×/22 eyepiece and the 40× objective or approximately ≥100% of the FOV using the 60× objective as the criterion separating minimal from mild severities.

Perfluorooctane Sulfonate-Induced Hepatic Steatosis in Male Sprague Dawley Rats Is Not Attenuated by Dietary Choline Supplementation.

Perfluorooctane sulfonate (PFOS) is an environmentally persistent chemical. Dietary 100 ppm PFOS fed to male mice and rats for 4 weeks caused hepatic steatosis through an unknown mechanism. Choline deficient diets can cause hepatic steatosis. A hepatic choline:PFOS ion complex was hypothesized to cause this effect in mice. This study tested whether dietary choline supplementation attenuates PFOS-induced hepatic steatosis in rats. Sprague Dawley rats (12/sex/group) were fed control, choline supplemented (CS), 100 ppm PFOS, or 100 ppm PFOS + CS diets for 3 weeks. Male rats fed both PFOS-containing diets had decreased serum cholesterol and triglycerides (TGs) on days 9, 16, and/or 23 and increased hepatic free fatty acids and TG (ie, steatosis). Female rats fed both PFOS diets had decreased serum cholesterol on days 9 and 16 and decreased hepatic free fatty acid and TG at termination (ie, no steatosis). Liver PFOS concentrations were similar for both sexes. Liver choline concentrations were increased in male rats fed PFOS (±CS), but the increase was lower in the PFOS + CS group. Female liver choline concentrations were not altered by any diet. These findings demonstrate a clear sex-related difference in PFOS-induced hepatic steatosis in the rat. Additional evaluated mechanisms (ie, nuclear receptor activation, mRNA upregulation, and choline kinase activity inhibition) did not appear to be involved in the hepatic steatosis. Dietary PFOS (100 ppm) induced hepatic steatosis in male, but not female, rats that was not attenuated by choline supplementation. The mechanism of lipid accumulation and the sex-related differences warrant further investigation.

Acute and Chronic Kidney Injury in a Non-Human Primate Model of Partial-Body Irradiation with Bone Marrow Sparing.

The development of medical countermeasures against acute and delayed multi-organ injury requires animal models predictive of the human response to radiation and its treatment. Late chronic injury is a well-known feature of radiation nephropathy, but acute kidney injury has not been reported in an appropriate animal model. We have established a single-fraction partial-body irradiation model with minimal marrow sparing in non-human primates. Subject-based medical management was used including parenteral fluids according to prospective morbidity criteria. We show herein that 10 or 11 Gy exposures caused both acute and chronic kidney injury. Acute and chronic kidney injury appear to be dose-independent between 10 and 11 Gy. Acute kidney injury was identified during the first 50 days postirradiation and appeared to resolve before the occurrence of chronic kidney injury, which was progressively more severe up to 180 days postirradiation, which was the end of the study. These findings show that mitigation of the acute radiation syndrome by medical management will unmask delayed late effects that occur months after partial-body irradiation. They further emphasize that both acute and chronic changes in kidney function must be taken into account in the use and timing of mitigators and medical management for acute radiation syndrome and delayed effects of acute radiation exposure (DEARE).

Four-week dietary supplementation with 10- and/or 15-fold basal choline caused decreased body weight in Sprague Dawley rats.

Choline is an essential nutrient utilized for phosphatidylcholine biosynthesis and lipoprotein packaging and secretion. Recently, choline supplementation has been used by athletes and the public for weight loss. However, the potential toxicological impact of choline dietary supplementation requires further investigation. This study examined the effects of choline dietary supplementation in Sprague Dawley rats for 4 weeks. Rats were fed diets containing basal choline levels (control) or 5-, 10-, or 15-fold (5×, 10×, or 15×) basal diet concentration. In groups fed choline-supplemented diets, there were no toxicologically relevant findings in clinical observations, food intake, clinical chemistry, liver weights, or liver histopathology. However, decreased mean body weights (8.5-10.2%) and body weight gains (24-31%) were noted for the 10× choline-supplemented (females only) and 15× choline-supplemented (both sexes) groups relative to the control groups from day 3 onward. These body weight effects were not related to a persistent reduction in average food intake. Serum cholesterol was increased in the 15× choline-supplemented male rats relative to the controls, an expected effect of choline supplementation; however, there were no changes in the serum cholesterol of female rats. Serum choline concentrations were increased in female rats relative to the male rats across all treatment groups. The maximum tolerated dose for male and female rats were the 15× and 10× choline supplements, respectively, based on decreased mean body weight and body weight gains. This study supported the conclusions of a clinical trial that showed a high choline diet can decrease body weight in humans.

Postnatal Organ Development as a Complicating Factor in Juvenile Toxicity Studies in Rats.

Toxicologic pathologists must evaluate tissues of immature animals from a number of types of nonclinical toxicity studies. The pathologist who is familiar with normal postnatal organ development is in a better position to appropriately detect and differentiate between abnormal, delayed, or precocious development. Vacuolation and apoptosis in multiple tissue types are normal components of development that could influence the interpretation of some tissues. Unique postnatal features such as the germal matrix in the brain, gonocytes in the testes, and saccules in the lung may complicate the histopathological evaluation. With the knowledge of normal organ development and critical windows therein, it is possible to design targeted studies to identify xenobiotic toxicity.