Development and validation of an ultraperformance liquid chromatography-tandem mass spectrometry method for quantitation of total 3,3',5-triiodo-L-thyronine and 3,3',5,5'-tetraiodo-L-thyronine in rodent serum.

Many environmental chemicals are known to disrupt thyroid function. Measurement of thyroid hormones in animal studies provides useful information to understand the effects of environmental chemicals on thyroid hormone metabolism. We report an efficient method, utilizing a protein precipitation followed by ultraperformance liquid chromatography-tandem mass spectrometry analysis, to quantitate total 3,3',5-triiodo-L-thyronine (triiodothyronine, T3) and total 3,3',5,5'-tetraiodo-L-thyronine (thyroxine, T4) in rodent serum. The use of synthetic serum for calibration standards eliminated the interferences from endogenous total T3 and T4 and allowed the experimental lower limits of quantitation (LOQ) to be set at the required concentration (T3, 20 ng/dL; T4, 0.5 µg/dL) to allow quantitation of endogenous concentrations. The method was linear (r>0.99; range 20.0-600 ng/dL T3, 0.500-15 µg/dL T4) with good assay recoveries (90.4-107%) for both analytes. Intra- and inter-day accuracy, estimated as percent relative error, were ≤ ±7.6% and intra- and inter-day precision, estimated as the relative standard deviation, were ≤ 5.3% for both analytes. The method may easily be adapted to a well-plate format thereby further improving the efficiency. Total T3 and T4 concentrations were stable in male and female rat and mouse serum when stored in the freezer (~ -70 °C) for up to 62 d with determined values within 92.8-111% of day 0 for both analytes. The method can be extended to quantitate total T3 and T4 concentrations in humans or other species with minimal optimization.

Immunotoxicity studies of sulfolane following developmental exposure in Hsd:Sprague Dawley SD rats and adult exposure in B6C3F1/N mice.

Sulfolane is a solvent used in the petrochemical industry and a groundwater contaminant in areas near refineries. The current studies were conducted to assess the impact of oral exposure to sulfolane on the immune system using two models: (1) a perinatal drinking water exposure to 0, 30, 100, 300, or 1000 mg/L from gestation day (GD) 6 until ∼13 weeks-of-age in Harlan Sprague Dawley rats; and, (2) a 90-day gavage exposure of adult female B6C3F1/N mice to 0, 1, 10, 30, 100, or 300 mg/kg/day. Immune parameters evaluated included measurement of antibody production against sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), ex vivo measurements of natural killer (NK) cell activity, cytotoxic T-cell (CTL) activity, and T-cell proliferation, as well as measures of splenic immune cell populations, hematological parameters, and histopathology of immune tissues. A decrease in ex vivo NK cell activity was observed in cells from female - but not male - F1 rats following developmental exposure. In adult female mice, splenic NK cell number was lower than the vehicle controls at doses ≥ 100 mg/kg; however, ex vivo NK cell activity was not affected by sulfolane treatment. In female mice, a decrease in the number of large unstained cells at doses ≥ 30 mg/kg was observed. In F1 rats, effects on white blood cells (WBC) were limited to a decreasing trend in leukocytes in females; no effects were observed in males. Under the conditions of this study, a no-observed-effect level (NOEL) of 3 mg/kg/day was identified based on reduced NK cell activity in female F1 rats. Overall, these findings suggest that oral exposure to sulfolane in rodents had minimal effects on the immune system.

Transcriptomic data from the rat liver after five days of exposure to legacy or emerging brominated flame retardants.

Large-scale gene expression analysis of legacy* and emerging** brominated flame retardants were conducted in the male Harlan Sprague Dawley rat [1]. Each animal was dosed for 5 days with the chemical at concentrations of 0.1 - 1000 µmol/kg body weight per day. Following the last dose, a specimen of the left liver was removed for RNA extraction. The amplified RNA (aRNA) was fragmented and then hybridized to Affymetrix Rat Genome 230 2.0 Arrays. Each GeneChip® array was scanned using an Affymetrix GeneChip® Scanner 3000 7 G to generate raw expression level data (.CEL files). Statistical contrasts were used to find pairwise gene expression differences between the control group and each dose group using the R/maanova package [2]. The transcriptomic data can be used to provide insights into the degree of toxicity, toxic mechanisms, disease pathways activated by exposure, and for benchmark dose analysis. The gene expression data for each of the nine flame retardants discussed here accompanies the research article entitled, "Comparative Toxicity and Liver Transcriptomics of Legacy and Emerging Brominated Flame Retardants following 5-Day Exposure in the Rat" [1]. * polybrominated diphenyl ether 47 (PBDE 47), decabromodiphenyl ether (decaBDE), hexabromocyclododecane (HBCD); ** 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB); bis(2-ethylhexyl) tetrabromophthalate (TBPH); tetrabromobisphenol A-bis(2,3-dibromopropyl ether (TBBPA-DBPE); 1,2-bis(tribromophenoxy)ethane (BTBPE); decabromodiphenylethane (DBDPE); hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO).

Comparative toxicity and liver transcriptomics of legacy and emerging brominated flame retardants following 5-day exposure in the rat.

The relative toxicity of three legacy and six emerging brominated flame retardants* was studied in the male Harlan Sprague Dawley rat. The hepatocellular and thyroid toxicity of each flame retardant was evaluated following five-day exposure to each of the nine flame retardants (oral gavage in corn oil) at 0.1-1000 µmol/kg body weight per day. Histopathology and transcriptomic analysis were performed on the left liver lobe. Centrilobular hypertrophy of hepatocytes and increases in liver weight were seen following exposure to two legacy (PBDE-47, HBCD) and to one emerging flame retardant (HCDBCO). Total thyroxine (TT4) concentrations were reduced to the greatest extent after PBDE-47 exposure. The PBDE-47, decaBDE, and HBCD liver transcriptomes were characterized by upregulation of liver disease-related and/or metabolic transcripts. Fewer liver disease or metabolic transcript changes were detected for the other flame retardants studied (TBB, TBPH, TBBPA-DBPE, BTBPE, DBDPE, or HCDBCO). PBDE-47 exhibited the most disruption of hepatocellular toxic endpoints, with the Nrf2 antioxidant pathway transcripts upregulated to the greatest extent, although some activation of this pathway also occurred after decaBDE, HBCD, TBB, and HCBCO exposure. These studies provide information that can be used for prioritizing the need for more in-depth brominated flame retardant toxicity studies.

Previously Diagnosed Reticulum Cell Hyperplasia in Decalcified Rat Bone Marrow Stain Positive for Ionized Calcium Binding Adapter Molecule 1 (Iba1): A Monocytic/Macrophage Cell Marker.

Reticulum cell hyperplasia (RCH) was a term used for many years by the National Toxicology Program (NTP) to describe a certain non-neoplastic bone marrow lesion of rats. Retrospective microscopic evaluation of RCH lesions and immunohistochemistry analyses were performed to reassess and further characterize these lesions. The NTP database was searched to identify femoral bone marrow specimens diagnosed with RCH from 1981 to 2014 (n = 254). The diagnosis last occurred in 2003, after which the term "cellular infiltration" was used. Eighty-three RCH slides, spanning 22 years, representing 34 different chemicals, were selected for microscopic review, and a subset (23) was chosen for ionized calcium binding adapter molecule 1 (Iba1) immunohistochemical staining; initial investigations revealed Iba1 worked as a macrophage marker on decalcified tissue. The following diagnoses were made upon reevaluation: 36 were consistent with cellularity increased, macrophage, 22 with histiocytic sarcoma, 8 with increased myeloid cells, 4 with autolysis, and 13 were normal appearance. All 23 RCH lesions stained positive for Iba1. Fifty-eight of 83 bone marrows previously diagnosed with RCH are consistent morphologically and immunohistochemically with cells of histiocytic origin. These results will help with interpretation of historical data and demonstrates that Iba1 can be used in decalcified bone marrow sections.

NTP/NIEHS Global Contributions to Toxicologic Pathology.

National Toxicology Program (NTP) pathologists are engaged in important initiatives that have significant global impact. These initiatives build on its leadership in pathology peer review and publications in the areas of toxicologic pathology, clinical pathology, and laboratory animal medicine. Over the past decade, NTP/National Institute of Environmental Health Sciences research initiatives have focused on cancer and noncancer hazard identification, with the goal of understanding cellular and molecular mechanisms of disease. New initiatives of significant global impact include the web-based nonneoplastic lesion atlas and an NTP partnership with international scientists to investigate molecular mechanisms at the whole genome level, which will be used to inform potential mechanisms of environmental exposures in human cancers. Also, we are dedicated to contributing to pathology and toxicology organizations through service on executive committees and editorial boards, participating in international projects and symposiums, and providing training for future leaders in toxicologic pathology. Herein, we provide highlights of our global contributions.

A Black Cohosh Extract Causes Hematologic and Biochemical Changes Consistent with a Functional Cobalamin Deficiency in Female B6C3F1/N Mice.

Black cohosh rhizome, available as a dietary supplement, is most commonly marketed as a remedy for dysmenorrhea and menopausal symptoms. A previous subchronic toxicity study of black cohosh dried ethanolic extract (BCE) in female mice revealed a dose-dependent ineffective erythropoiesis with a macrocytosis consistent with the condition known as megaloblastic anemia. The purpose of this study was to investigate potential mechanisms by which BCE induces these particular hematological changes. B6C3F1/N female mice (32/group) were exposed by gavage to vehicle or 1,000 mg/kg BCE for 92 days. Blood samples were analyzed for hematology, renal and hepatic clinical chemistry, serum folate and cobalamin, red blood cell (RBC) folate, and plasma homocysteine and methylmalonic acid (MMA). Folate levels were measured in liver and kidney. Hematological changes included decreased RBC count; increased mean corpuscular volume; and decreased reticulocyte, white blood cell, neutrophil, and lymphocyte counts. Blood smear evaluation revealed increased Howell-Jolly bodies and occasional basophilic stippling in treated animals. Plasma homocysteine and MMA concentrations were increased in treated animals. Under the conditions of our study, BCE administration caused hematological and clinical chemistry changes consistent with a functional cobalamin, and possibly folate, deficiency. Further studies are needed to elucidate the mechanism by which BCE causes increases in homocysteine and MMA.

Vaginal Cytology of the Laboratory Rat and Mouse: Review and Criteria for the Staging of the Estrous Cycle Using Stained Vaginal Smears.

Microscopic evaluation of the types of cells present in vaginal smears has long been used to document the stages of the estrous cycle in laboratory rats and mice and as an index of the functional status of the hypothalamic-pituitary-ovarian axis. The estrous cycle is generally divided into the four stages of proestrus, estrus, metestrus, and diestrus. On cytological evaluation, these stages are defined by the absence, presence, or proportion of 4 basic cell types as well as by the cell density and arrangement of the cells on the slide. Multiple references regarding the cytology of the rat and mouse estrous cycle are available. Many contemporary references and studies, however, have relatively abbreviated definitions of the stages, are in reference to direct wet mount preparations, or lack comprehensive illustrations. This has led to ambiguity and, in some cases, a loss of appreciation for the encountered nuances of dividing a steadily moving cycle into 4 stages. The aim of this review is to provide a detailed description, discussion, and illustration of vaginal cytology of the rat and mouse estrous cycle as it appears on smears stained with metachromatic stains.

Proceedings of the 2014 National Toxicology Program Satellite Symposium.

The 2014 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri" was held in Washington, D.C., in advance of the Society of Toxicologic Pathology's 33rd annual meeting. The goal of this annual NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting and discussion. Some lesions and topics covered during the symposium included a pulmonary mucinous adenocarcinoma in a male B6C3F1 mouse; plexiform vasculopathy in Wistar Han (Crl:WI[Han]) rats; staging of the estrous cycle in rats and mice; peri-islet fibrosis, hemorrhage, lobular atrophy and inflammation in male Sprague-Dawley (SD) rats; retinal dysplasia in Crl:WI[Han] rats and B6C3F1 mice; multicentric lymphoma with intravascular microemboli and tumor lysis syndrome, and 2 cases of myopathy and vascular anomaly in Tg.rasH2 mice; benign thymomas in Crl:WI[Han] rats; angiomatous lesions in the mesenteric lymph nodes of Crl:WI[Han] rats; an unusual foveal lesion in a cynomolgous monkey; and finally a series of nomenclatures challenges from the endocrine International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) Organ Working Group (OWG).

Proceedings of the 2013 National Toxicology Program Satellite Symposium.

The 2013 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri," was held in Portland, Oregon, in advance of the Society of Toxicologic Pathology's 32nd annual meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting and discussion. Some lesions and topics covered during the symposium included a caudal tail vertebra duplication in mice; nephroblastematosis in rats; ectopic C cell tumor in a hamster; granular cell aggregates/tumor in the uterus of a hamster; Pneumocystis carinii in the lung of a rat; iatrogenic chronic inflammation in the lungs of control rats; hepatoblastoma arising within an adenoma in a mouse; humoral hypercalcemia of benignancy in a transgenic mouse; acetaminophen-induced hepatotoxicity in rats; electron microscopy images of iatrogenic intraerythrocytic inclusions in transgenic mice; questionable hepatocellular degeneration/cell death/artifact in rats; atypical endometrial hyperplasia in rats; malignant mixed Müllerian tumors/carcinosarcomas in rats; differential diagnoses of proliferative lesions of the intestine of rodents; and finally obstructive nephropathy caused by melamine poisoning in a rat.

Toxicity and carcinogenicity studies of Ginkgo biloba extract in rat and mouse: liver, thyroid, and nose are targets.

Ginkgo biloba extract (GBE) is a popular herbal supplement that is used to improve circulation and brain function. In spite of widespread human exposure to relatively high doses over potentially long periods of time, there is a paucity of data from animal studies regarding the toxicity and carcinogenicity associated with GBE. In order to fill this knowledge gap, 3-month and 2-year toxicity and carcinogenicity studies with GBE administered by oral gavage to B6C3F1/N mice and F344/N rats were performed as part of the National Toxicology Program's Dietary Supplements and Herbal Medicines Initiative. The targets of GBE treatment were the liver, thyroid, and nose. These targets were consistent across exposure period, sex, and species, albeit with varying degrees of effect observed among studies. Key findings included a notably high incidence of hepatoblastomas in male and female mice and evidence of carcinogenic potential in the thyroid gland of both mice and rats. Various nonneoplastic lesions were observed beyond control levels in the liver, thyroid gland, and nose of rats and mice administered GBE. Although these results cannot be directly extrapolated to humans, the findings fill an important data gap in assessing risk associated with GBE use.

Comparison of automated versus manual neutrophil counts for the detection of cellular abnormalities in dogs receiving chemotherapy: 50 cases (May to June 2008).

OBJECTIVE: To determine the frequency of clinically relevant abnormalities missed by failure to perform a blood smear evaluation in a specific subset of dogs receiving chemotherapy and to compare automated and manual neutrophil counts in the same population. DESIGN: Retrospective case series. ANIMALS: 50 dogs receiving chemotherapy with a total nucleated cell count > 4,000 nucleated cells/µL. PROCEDURES: 50 blood smears were evaluated for abnormalities that have strong potential to change the medical plan for a patient: presence of blast cells, band neutrophils, nucleated RBCs, toxic change, hemoparasites, schistocytes, and spherocytes. Automated and manual neutrophil counts were compared. RESULTS: Blood smears from 10 (20%) patients had ≥ 1 abnormalities. Blast cells were identified on 4 (8%) blood smears, increased nucleated RBCs were identified on 5 (10%), and very mild toxic change was identified on 2 (4%). Correlation coefficient of the neutrophil counts was 0.96. Analysis revealed a slight bias between the automated and manual neutrophil counts (mean ± SD difference, -0.43 × 10(3)/µL ± 1.10 × 10(3)/µL). CONCLUSIONS AND CLINICAL RELEVANCE: In this series of patients, neutrophil count correlation was very good. Clinically relevant abnormalities were found on 20% of the blood smears. An automated CBC appears to be accurate for neutrophil counts, but a microscopic examination of the corresponding blood smear is still recommended; further studies are needed to determine whether the detection or frequency of these abnormalities would differ dependent on chemotherapy protocol, neoplastic disease, and decision thresholds used by the oncologist in the ordering of a CBC without a blood smear evaluation.

Effect of a combined aspiration and core biopsy technique on quality of core bone marrow specimens.

For cases requiring both a bone marrow aspirate and core biopsy, using the same needle and site (i.e., a combined technique) can decrease time, expense, and discomfort compared with the independent (direct) collection of each specimen. The benefits of the combined approach should not be achieved at the expense of specimen quality. In this study, core bone marrow samples obtained from the proximal humerus of 26 dogs by a combined technique immediately posteuthanasia were compared with core samples obtained directly from the opposite humerus. Both core samples from each dog were compared for relative overall quality. Biopsies were unsuccessful in four dogs using the combined technique and in one dog by the direct technique. Marrow length was shorter and hemorrhage artifact was more common using the combined technique. There were no differences in cellularity, megakaryocyte count, the myeloid/erythroid ratio (M/E ratio), iron stores, or diagnostic quality. Direct core biopsy remains the ideal technique; however, the degree of compromise was small in the samples obtained from the combined technique. For clinical patients, the advantages of the combined technique must continue to be weighed against potential loss of diagnostic sensitivity.

Proceedings of the 2012 National Toxicology Program Satellite Symposium.

The 2012 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri," was held in Boston in advance of the Society of Toxicologic Pathology's 31st annual meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting or discussion. Some lesions and topics covered during the symposium include eosinophilic crystalline pneumonia in a transgenic mouse model; differentiating adrenal cortical cystic degeneration from adenoma; atypical eosinophilic foci of altered hepatocytes; differentiating cardiac schwannoma from cardiomyopathy; diagnosis of cardiac papillary muscle lesions; intrahepatocytic erythrocytes and venous subendothelial hepatocytes; lesions in Rathke's cleft and pars distalis; pernicious anemia and megaloblastic disorders; embryonic neuroepithelial dysplasia, holoprosencephaly and exencephaly; and INHAND nomenclature for select cardiovascular lesions.

An ethanolic extract of black cohosh causes hematological changes but not estrogenic effects in female rodents.

Black cohosh rhizome (Actaea racemosa) is used as a remedy for pain and gynecological ailments; modern preparations are commonly sold as ethanolic extracts available as dietary supplements. Black cohosh was nominated to the National Toxicology Program (NTP) for toxicity testing due to its widespread use and lack of safety data. Several commercially available black cohosh extracts (BCE) were characterized by the NTP, and one with chemical composition closest to formulations available to consumers was used for all studies. Female B6C3F1/N mice and Wistar Han rats were given 0, 15 (rats only), 62.5 (mice only), 125, 250, 500, or 1000 mg/kg/day BCE by gavage for 90 days starting at weaning. BCE induced dose-dependent hematological changes consistent with a non-regenerative macrocytic anemia and increased frequencies of peripheral micronucleated red blood cells (RBC) in both species. Effects were more severe in mice, which had decreased RBC counts in all treatment groups and increased micronucleated RBC at doses above 125 mg/kg. Dose-dependent thymus and liver toxicity was observed in rats but not mice. No biologically significant effects were observed in other organs. Puberty was delayed 2.9 days at the highest treatment dose in rats; a similar magnitude delay in mice occurred in the 125 and 250 mg/kg groups but not at the higher doses. An additional uterotrophic assay conducted in mice exposed for 3 days to 0.001, 0.01, 0.1, 1, 10, 100 and 500 mg/kg found no estrogenic or anti-estrogenic activity. These are the first studies to observe adverse effects of BCE in rodents.

Artifactual changes in Sprague-Dawley rat hematologic parameters after storage of samples at 3 °C and 21 °C.

Circumstances can occur that prevent timely analysis of blood samples. The purpose of this study was to characterize artifactual changes in rat hematologic parameters after storage of samples at 3 and 21 °C and to document the effects of storage on peripheral blood smear findings. EDTA-treated blood samples were collected from 12 male Sprague-Dawley rats. Samples were analyzed on an impedance hematology analyzer within 5 min after collection and then at 6, 24, 48, and 72 h after storage at 3 °C or 21 °C. Corresponding blood smears were examined microscopically. RBC count and hemoglobin concentration had not changed after 72 h at either temperature. At 3 °C, the instrument-derived hematocrit and manually measured PCV remained unchanged for 72 h. Compared with 0-h values, platelet counts and MCV at 6 h and MPV at 24 h were higher at either temperature. In general, WBC count and neutrophil and lymphocyte percentages were unchanged for at least 48 h at either temperature. Prominent blood smear findings were smudge cells, pyknotic leukocytes, echinocytes, and spheroechinocytes. Although some observed changes were within analytic variability or clinically negligible, the best practice likely is to measure hematologic parameters within 6 h after collection. In the event of delayed analysis, specimens should be stored in the refrigerator, and care must be taken not to misinterpret artifactual changes as pathologic findings.